Compositions and methods for increasing serum half-life

ABSTRACT

Provided herein are glycovariant Fc fusion proteins having increased serum half lives. Also provided are methods for increasing the serum half life of an Fc fusion protein by introducing one or more non-endogenous glycosylation sites.

RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 61/494,866, filed Jun. 8, 2011. The specificationof the foregoing application is hereby incorporated by reference in itsentirety.

BACKGROUND

Therapeutic proteins or peptides in their native state, or whenrecombinantly produced, are typically labile molecules exhibiting shortperiods of stability or short serum half-lives. In addition, thesemolecules are often extremely labile when formulated, particularly whenformulated in aqueous solutions for diagnostic and therapeutic purposes.Few practical solutions exist to extend or promote the stability in vivoor in vitro of proteinaceous therapeutic molecules. Many therapeutics,in particular peptide drugs, suffer from inadequate serum half-lives invivo. This necessitates the administration of such therapeutics at highfrequencies and/or higher doses, or the use of sustained releaseformulations, in order to maintain the serum levels necessary fortherapeutic effects. Frequent systemic administration of drugs isassociated with considerable negative side effects. For example,frequent, e.g. daily, systemic injections represent a considerablediscomfort to the subject, pose a high risk of administration relatedinfections, and may require hospitalization or frequent visits to thehospital, in particular when the therapeutic is to be administeredintravenously. Moreover, in long-term treatments daily intravenousinjections can also lead to considerable tissue scarring and vascularpathologies caused by the repeated puncturing of vessels. Similarproblems are known for all frequent systemic administrations oftherapeutics, for example, the administration of insulin to diabetics,or interferon drugs in patients suffering from multiple sclerosis. Allthese factors lead to a decreased patient compliance and increased costsfor the health system.

One possible solution to modify serum half-life of a pharmaceuticalagent is to covalently attach to the agent molecules that may increasethe half-life. Previously, it has been shown that attachment ofpolymers, such as polyethylene glycol or “PEG”, to polypeptides mayincrease their serum half-lives. However, the attachment of polymers canlead to decreases in drug activity. Incomplete or non-uniform attachmentleads to a mixed population of compounds having differing properties.Additionally, the changes in half-lives resulting from suchmodifications are unpredictable. For example, conjugation of differentpolyethylene glycols to IL-8, G-CSF and IL-1ra produced molecules havinga variety of activities and half-lives (Gaertner and Offord, (1996),Bioconjugate Chem. 7:38-44). Conjugation of IL-8 to PEG₂₀ produced nochange in its half-life, while conjugation of PEG₂₀ to IL-1ra gave analmost seven-fold increase in half-life. Additionally, the IL-8/PEG₂₀conjugate was ten- to twenty-fold less effective than the nativeprotein.

Accordingly, methods that are capable of increasing the serum half-lifeof a biologically active molecule, without seriously diminishing thebiological function of the molecule, would be highly desirable.

SUMMARY

In part, the disclosure provides methods for extending the serumhalf-life of a fusion protein comprising an immunoglobulin Fc domain andat least one heterologous polypeptide domain. In certain embodiments,the methods include preparing a modified nucleic acid encoding amodified Fc fusion protein that has an extended serum half-life relativeto an initial Fc fusion protein by modifying the nucleic acid encodingthe heterologous portion of the initial Fc fusion protein to code forone or more additional N-linked glycosylation sites. In someembodiments, the modified nucleic acid of the invention will encode amodified Fc fusion protein that, when expressed in a suitable cellculture, has a serum half-life at least 10% longer than the serumhalf-life of the initial Fc fusion protein. In some embodiments, themodified Fc fusion protein of the invention has substantially the sameor greater in vivo biological activity relative to the unmodified Fcfusion protein. In certain embodiments, glycosylation at one or more ofthe additional (i.e., introduced) glycosylation sites increases thehalf-life (e.g., in vitro, in vivo, serum half-life) of the modified Fcfusion protein by at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 125%,150%, 175%, 200%, 225%, or 250% or more relative to the serum half-lifeof a fusion polypeptide lacking the additional glycosylation site. Incertain embodiments, serum half-life of a modified Fc fusion protein anda fusion polypeptide lacking the additional glycosylation site aremeasured in the same animal model or species for comparison. Inexemplary embodiments, the serum half-life is measured in apharmacokinetic rat assay or a pharmacokinetic monkey assay as describedherein.

In some embodiments, the disclosure provides a method for preparing amodified Fc fusion protein that has an extended half-life relative to aninitial Fc fusion protein by a) expressing a nucleic acid that has beenmodified to introduce at least one additional N-linked glycosylationsite in a cell culture that provides mammalian or mammalian-likeglycosylation, and b) recovering the modified Fc fusion protein from thecell culture. Fc fusion proteins may be recovered as crude, partiallypurified, or highly purified fractions using any technique suitable forobtaining protein from cell cultures. In certain aspects the modifiednucleic acid is expressed in a cell line that generates N-linked sugarmoieties that comprise sialic acid. In certain aspects, the modifiednucleic acid is expressed by a mammalian cell line, including but notlimited to, a CHO cell line, a NSO cell line, a COS cell line, or aHEK236 cell line. In other aspects, the modified nucleic acid isexpressed by a non-mammalian cell that has been engineered to providemammalian or mammalian-like glycosylation, including but not limited to,genetically engineered fungal cells, insect cells, or plant cells. Incertain aspects, purification may include the steps of exposing modifiedFc fusion protein to protein A and recovering the modified Fc fusionprotein that is bound to the protein A. In preferred embodiments, the Fcfusion proteins produced by the methods of the disclosure are formulatedfor administration to a patient.

In some embodiments, the disclosure provides a cell line comprising amodified nucleic acid prepared to according any of the methods describedherein. A cell line of the invention may include a mammalian cell line(e.g., CHO cell line, a NSO cell line, a COS cell line, or a HEK236 cellline) or a non-mammalian cell line (e.g., fungal cells, insect cells, orplant cells), which has been genetically modified to provide mammalianor mammalian-like glycosylation.

In certain embodiments, the disclosure provides glycovariant Fc fusionproteins characterized by increased stability and/or serum half life andmethods for producing fusion proteins having increased half-lives.Fc-fusion proteins of the invention include, but are not limited to,polypeptides comprising an immunoglobulin Fc domain and at least oneheterologous polypeptide domain. The Fc-fusion proteins are modifiedoutside of the immunoglobulin Fc domain to introduce at least onenon-endogenous N-linked glycosylation site, which increases the serumhalf-life of the modified fusion protein relative to the half-life(e.g., in vitro, in vivo, or serum half-life) of the fusion proteinlacking the introduced glycosylation site. In certain embodiments, afusion protein of the invention comprise at least one non-endogenous, orintroduced, N-linked glycosylation site that increases the serumhalf-life of the fusion by at least 10%, 20%, 30%, 40%, 50%, 75%, 100%,125%, 150%, 175%, 200%, 225%, or 250% or more relative to the serumhalf-live of a fusion protein lacking the introduced glycosylation site.In certain embodiments, serum half-life of a modified Fc fusion proteinand a fusion polypeptide lacking the additional glycosylation site aremeasured in the same animal model or species for comparison. Inexemplary embodiments, the serum half-life is measured in apharmacokinetic rat assay or a pharmacokinetic monkey assay as describedherein.

Fc fusion proteins of the invention may be modified by the addition,deletion, or substitution of one or more amino acid residues tointroduce one N-linked glycosylation site. In certain embodiments, theheterologous portion of the initial Fc fusion protein comprises at leastone N-linked glycosylation site per each 50, 55, 60, 65, 70, 75, 80, 85,90, 95 100, 105 110, 115, 120, 130, 140, or 150 amino acids. In certainembodiments, the heterologous portion of the initial Fc fusion proteincomprises fewer than one N-linked glycosylation site per each 60, 70,80, 90, 100, 110, 120, or 125 amino acids. In certain embodiments, eachamino acid of the heterologous portion of the modified Fc fusion proteinthat is attached to an N-linked glycosylation is separated by at least15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids from any otheramino acid modified by an N-linked glycosylation. In certainembodiments, the additional N-linked glycosylation site does not occurwithin 10, 20, 30, 40, or 50 amino acids of the N-terminus, C-terminus,or both N-and C-termini of the modified Fc fusion protein.

In preferred embodiments, Fc fusion proteins of the invention comprisesat least two structurally distinct domains that are connected by anamino acid sequence that is surface exposed (i.e., a soluble loopdomain) and not incorporated into an α-helix or β-sheet. In preferredembodiments, additional N-linked glycosylation sites are positionedwithin amino acid sequences that are surface exposed. In preferredembodiments, additional N-linked glycosylation sites are notincorporated into a region of the protein having a secondary structuralelement, e.g., an α-helix or β-sheet. Heterologous portions of use inthe instant invention may include a functional domain of a protein(e.g., an enzymatic or catalytic domain or ligand binding domain). Insome embodiments, the Fc fusion protein of the disclosure furthercomprises a linker domain. In some embodiments, at least one of theintroduced glycosylation sites is located in the linker domain. Aheterologous domain of a fusion polypeptide of the invention ispreferably of higher molecular weight, being at least 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 kDa.

In specific embodiments, the heterologous portion comprises anextracellular (i.e., soluble) domain of a cellular receptor (e.g.,transmembrane receptor) as well as any variants thereof (includingmutants, fragments, and peptidomimetic forms). In preferred embodiments,the heterologous portion includes a ligand binding domain of atransmembrane receptor. Methods of the disclosure may be used tointroduce one or more N-linked glycosylation sites at positions of theheterologous domain such that any additional sugar moieties attached donot substantially interfere with the ligand binding domain, e.g., lessthan a 2-, 3-, 5-, 10-, or 15-fold reduction in binding activityrelative to ligand binding to the Fc-fusion protein lacking theintroduced glycosylation site. Therefore, in some aspects, one or moreof the introduced N-linked glycosylation sites are introduced atpositions such that the amino acid sequence of the ligand binding domainis not modified and/or at positions that are predicted not to interferesubstantially with the ligand interface. In certain embodiments, the Fcfusion protein has an IC₅₀ (i.e., half maximal inhibitory concentration)that is no more than two-, three-, five- or ten-fold less than that ofthe initial Fc fusion protein.

In certain embodiments, serum half-life of hyperglycosylated proteinsdescribed herein may be improved by the deletion of C- and/or N-terminalresidues of a protein domain, such as the extracellular domain of areceptor (e.g., TNFR2 or CTLA4). Preferably, such deletions are designedso as not to disrupt any secondary structure elements, such asalpha-helices or beta-sheets. Deletions may be 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 21, 25, 30 or more amino acids at the N- and/orC-terminus.

In certain embodiments, the Fc-fusion protein comprises a heterologousportion selected from a member of the nerve growth factor/tumor necrosisfactor receptor family. In specific embodiments, the extracellularreceptor domain comprises a portion of the tumor necrosis factor type 2receptor (i.e., TNFR2). In preferred embodiments, the disclosureprovides TNFR2 fusion proteins that bind to TNF-α with a K_(D) less than1 micromolar or less than 100, 10 or 1 nanomolar. In some embodiments,TNFR2 fusion proteins of invention comprise a TNFR2 extracellular domainhaving one or more modified amino acids located at positions D47, Q48,A50, E155, or G253 of the human TNFR2 precursor protein (i.e., SEQ IDNO: 1). These residues correspond to positions D25, Q26, A28, E133, andG231, respectively, of the extracellular, processed domain of TNFR2(i.e., SEQ ID NO: 2). Preferred modification include addition,substitution, and/or deletion of amino acids that result in theintroduction of one or more N-linked glycosylation sites in a TNFR2polypeptide including, for example, amino acid substitutions Q26N, A28S(or A28T), D25N, E133N, D25N, G231N relative to the amino acid sequenceof SEQ ID NO: 2. In some embodiments, more than one modification (e.g.,additions, deletions, and/or substitutions) are made to a TNFR2 fusionprotein to introduce one or more N-linked glycosylation sites including,for example, a TNFR2 variant comprising Q26N/A28S; Q26N/A28T;D25N/E133N; D25N/G231N; Q26N/A28S/E133N; Q26N/A28T/E133N;Q26N/A28S/G231N; Q26N/A28T/G231N; or E133N/G231N substitutions relativeto the amino acid sequence of SEQ ID NO: 2 or 44. A TNFR2-Fc fusionprotein of the invention may be any of those disclosed herein, such as afusion protein comprising a polypeptide having an amino acid sequenceselected from SEQ ID NOs: 1, 2, 5, 6, 7, 8, 16, 44, 46, 47, 53 or 54 orcomprising an amino acid sequence that is at least 80%, 85%, 90%, 95%,97% or 99% identical to an amino acid sequence selected from SEQ ID NOs:1, 2, 5, 6, 7, 8, 16, 44, 46, 47, 53 or 54. TNFR2-Fc fusion proteins maybe formulated as a pharmaceutical preparation comprising the TNFR2-Fcfusion protein and a pharmaceutically acceptable carrier, wherein thepreparation is substantially free of pyrogenic materials so as to besuitable for administration of a mammal. The composition may be at least95% pure, with respect to other polypeptide components, as assessed bysize exclusion chromatography, and optionally, the composition is atleast 98% pure.

In certain embodiments, the Fc-fusion protein comprises a heterologousportion of the cytotoxic T-lymphocyte antigen 4 (CTLA4) receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the amino acid sequence of a TNFR2-h(1)Fc polypeptide (SEQID NO: 5). A soluble TNFR2 fusion protein is depicted having theextracellular domain of human TNFR2 fused without linker to animmunoglobulin Fc domain, designated the h(1)Fc variant. Theimmunoglobulin Fc region is underlined once and the naturally occurringN-linked glycosylation sites are double underlined.

FIG. 2 shows the amino acid sequence of a Q48N/A50S variant TNFR2-h(1)Fcpolypeptide (SEQ ID NO: 6). A soluble TNFR2 fusion protein is depictedhaving the extracellular domain of human TNFR2 fused without linker toan h(1)Fc immunoglobulin Fc domain. The naturally occurring TNFR2-Fcextracellular domain was modified at positions 48 and 50 to introduce anN-linked glycosylation site. The immunoglobulin Fc region is underlinedonce and the substituted amino acids are double underlined.

FIG. 3 shows the amino acid sequence of a variant (D47N/E155N)TNFR2-h(1)Fc polypeptide (SEQ ID NO: 7). A soluble TNFR2 fusion proteinis depicted having the extracellular domain of human TNFR2 fused withoutlinker to an h(1)Fc immunoglobulin Fc domain. The naturally occurringTNFR2-Fc extracellular domain was modified at positions 47 and 155 tointroduce an N-linked glycosylation site. The immunoglobulin Fc regionis underlined once and the substituted amino acids are doubleunderlined.

FIG. 4 shows the amino acid sequence of a variant (D47N/G253N)TNFR2-h(1)Fc polypeptide (SEQ ID NO: 8). A soluble TNFR2 fusion proteinis depicted having the extracellular domain of human TNFR2 fused withoutlinker to an h(1)Fc immunoglobulin Fc domain. The naturally occurringTNFR2-Fc extracellular domain was modified at positions 47 and 253 tointroduce an N-linked glycosylation site. The immunoglobulin Fc regionis underlined once and the substituted amino acids double underlined.

FIG. 5 shows the nucleic acid sequence encoding the TNFR2-h(1)Fcpolypeptide (i.e., SEQ ID NO:5) with a natural leader sequence (SEQ IDNO: 9).

FIG. 6 is a ribbon diagram depicting the binding interface between TNFand the extracellular domain of the TNFR2 receptor. TNF is the greenribbon structure on the left, while the TNFR2 receptor is blue ribbonstructure on the right. Engineered sites to introduce N-linkedglycosylation sites are shown in circles. Single mutations arerepresented by blue circles and double mutations are represented bygreen circles. Mutations that significantly reduced ligand bindingactivity are located proximal to the TNF binding site and are depictedas red circles.

FIG. 7 shows the sequence of the extracellular domain of TNFR2 (SEQ IDNO:26) with exemplary amino acid modification for introducingnon-endogenous glycosylation sites being highlighted (SEQ ID NOS: 27-42,respectively, in order of appearance). Specific examples ofmodifications that would introduce a non-endogenous glycosylation siteare indicated for each highlighted region. The signal peptide isindicated with a single underline, and endogenous glycosylation sitesare indicated with a double underline.

FIG. 8 shows the predicted location of beta strands (arrows) in theTNFR2 structure (SEQ ID NO:43). Numbering is based on the native TNFR2precursor sequence (SEQ ID NO: 1). Secondary structure was inferred bysequence comparison and structural homology with TNFR1 (PDB ID: 1EXT),cytokine response modifier E (CrmE; PDB ID: 2UWI), and CD134 (OX40; PDBID: 2HEY).

FIG. 9 shows the amino acid sequence of a variant TNFR2-h(2)Fcpolypeptide (SEQ ID NO: 16) with a native leader sequence. A solubleTNFR2 fusion protein is depicted having the extracellular domain ofnative human TNFR2 fused via a four-amino-acid linker to an exemplaryhuman Fc domain (SEQ ID NO: 14). The Fc domain is indicated with asingle underline, and the leader and linker sequences are doubleunderlined.

FIG. 10 shows a nucleotide sequence (SEQ ID NO: 17) encoding theTNFR2-h(2)Fc polypeptide (i.e., SEQ ID NO: 16). The sequence encodingthe Fc domain is indicated with a single underline, and sequencesencoding the leader and linker are double underlined.

FIG. 11 shows the effect of glycovariant Q48N/A50S TNFR2-h(1)Fc on pawvolume as a marker of inflammation in a rat collagen-induced-arthritismodel. Collagen was administered on Days 0 and 7, whereas treatment withtest articles began on Day 6. Data shown are means±SEM. LikeTNFR2-h(1)Fc, treatment with Q48N/A50S TNFR2-h(1)Fc prevented the pawswelling observed in vehicle-treated rats during the second half of thestudy.

FIG. 12 shows the effect of glycovariant Q48N/A50S TNFR2-h(1)Fc on bonequality in a rat collagen-induced-arthritis model. Tarsal images wereobtained ex vivo by micro-computed tomography (micro-CT) after studycompletion on Day 21. Like TNFR2-h(1)Fc, treatment with Q48N/A50STNFR2-h(1)Fc prevented the bone erosion observed in vehicle-treatedrats.

DETAILED DESCRIPTION 1. Overview

In certain aspects, the present disclosure relates to the surprisingdiscovery that the serum half-life of an Fc-fusion protein can beextended by the introduction of at least one non-endogenous N-linkedglycosylation site at an appropriate position in an appropriate targetprotein. Accordingly, the disclosure provides methods for increasing theserum half-lives of Fc-fusions proteins, particular therapeuticpolypeptides, by increasing the number of N-linked glycosylation siteson the polypeptide. As demonstrated herein, the methods of thedisclosure have been used to increase the serum half-lives of Fc-fusionproteins comprising a portion of an extracellular domain of a receptorthat includes a ligand binding domain. In specific examples, thedisclosure provides modified TNFR2-Fc and CTLA4-Fc fusion proteins thatare characterized by an increased half-life relative to the unmodifiedforms of the respective fusion proteins. While not restricting thedisclosure to any particular mechanism of effect, it is proposed thatthe non-Fc portion of an Fc fusion protein (the “heterologous portion”)is exposed to a variety of intracellular and extracellular environmentsduring its residence time in a patient's body. These differingconditions may cause portions of the heterologous portion to becomevulnerable as substrates for proteases or other enzymes or moleculesthat begin the process of protein modification or degradation. Thus, thedisclosure provides a novel proposal that serum half-life of Fc fusionproteins is significantly affected by agents acting to degrade theheterologous portion, and that modifications that tend to protect theheterologous portion from such agents will lead to a greater serumhalf-life. As described herein, the addition of one or more N-linkedglycosylation sites provides a single-step, biocompatible system forshielding vulnerable portions of heterologous domains from unwanteddegradation or alteration and, in some instances, stabilizing desirablestructural conformations of such molecules.

It can be difficult to predict a priori which positions in aheterologous domain of an initial Fc fusion protein are most amenable toan additional N-linked glycosylation site, and in the absence of anyconstraints or guidelines any one heterologous domain couldtheoretically be modified with a nearly limitless combination of one ormore additional N-linked sites. Through work with different initial Fcfusion proteins, the applicants have found a variety of guidelines thatallow one to modify an initial Fc fusion protein at a reasonable numberof sites so as to arrive at a modified Fc fusion protein that exhibitsan extended serum half-life relative to the initial Fc fusion proteinwhile preserving biological activity (particularly in vivo biologicalactivity) of the modified molecule. While not wishing to limit the scopeof the overall disclosure, applicants have found that use of one or moreof the following principles provides a feasible approach to generatingan active, extended half-life Fc fusion protein: (1) N-linkedglycosylation sites may be advantageously placed at surface exposedpositions of the heterologous portion of an Fc fusion protein, whetherdetermined by protein structure analysis or empirical method, andparticularly at positions that are not contained within defined proteinstructure elements, such as alpha-helices or beta-sheets; (2) N-linkedglycosylation sites may be advantageously placed at positions in theheterologous portion of an Fc fusion protein that, if subject tomodification or degradation would cause substantial perturbation ofprotein structure or function (for example, many proteins have anunstructured region at the N-terminus that, if cleaved, causes little orno perturbation of the overall protein and its activity, andaccordingly, an N-linked glycosylation site placed at such a positionmay have modest effect on serum half-life, while by contrast surfaceexposed regions located between structure elements exhibit a combinationof high exposure and significant consequences to the protein if subjectto undesirable degradation and therefore surface exposed amino acidsbetween structure elements represent a desirable position for theintroduction of an N-linked glycosylation site); (3) N-linkedglycosylation sites may be placed at positions that do not interferewith a key functional site of the initial Fc fusion protein (e.g., aligand binding surface or catalytic site), which may be achieved byselecting positions external to the functional site itself and/orselecting positions where any new N-linked sugar moiety would notprotrude into the functional site; (4) the degree of effect to beachieved by the addition of an N-linked glycosylation site may beinversely proportional to the density of N-linked sugars already presentin the initial Fc fusion protein, and therefore, the approach may bemost effective with initial Fc fusion proteins that have a heterologousportion with a relatively low level of glycosylation (e.g., less than 1N-linked site per 60, 70, 80, 90, 100, 110, 125 or more amino acids ofthe heterologous portion); (5) in both heavily and lightly glycosylatedheterologous portions, the degree of effect to be achieved may beproportional to the spacing between N-linked glycosylation sites,meaning that one or more additional N-linked glycosylation sites may beadvantageously placed at a relatively regular and distant spacing (e.g.,greater than 10, 15, 20, 25, 30, 35 or 40 amino acids between the Nresidues of N-linked glycosylation sites) from existing N-linkedglycosylation or other additional N-linked glycosylation sites—even in aheavily glycosylated protein, if the sugars are closely clustered, theaddition of well-spaced N-linked sites may promote a significantincrease in serum half-life. While the use of one or more of the aboveprinciples may permit one to design a single modified Fc fusion proteinthat exhibits an extended serum half life relative to the initial Fcfusion protein, it will often be beneficial to generate a plurality ofmodified Fc fusion proteins based on a single initial Fc fusion protein,test these altered forms and then test combinations of those modifiedforms that exhibit the best combination of increased half-life andretained biological activity.

In certain aspects, the present disclosure relates to methods forincreasing the serum half-lives of Fc-fusions proteins, particulartherapeutic polypeptides, by increasing the number of N-linkedglycosylation sites on the polypeptide. As demonstrated herein, themethods of the disclosure have been used to increase the serumhalf-lives of Fc-fusion proteins comprising a portion of anextracellular domain of a receptor that includes a ligand bindingdomain. In specific examples, the disclosure provides modified TNFR2-Fcfusion proteins that are characterized by an increased half-liferelative to the unmodified forms of the respective fusion proteins.Regardless of the mechanism, it is apparent from the data presentedherein that introduction of additional glycosylation sites is aneffective method of prolonging the half-life of high molecular weightbiopharmaceuticals, in particular Fc-fusion proteins.

In specific embodiments, the disclosure provides modified TNFR2-Fcglycovariant fusion proteins characterized by increased serum half-life.Tumor Necrosis Factor (TNF) is a naturally occurring cytokine that isinvolved in normal inflammatory and immune responses. Tumor necrosisfactor-α (TNFα) and tumor necrosis factor-β (TNFβ) are homologousmultifunctional cytokines. The great similarities in structural andfunctional characteristics of these polypeptides have resulted in theircollective description as tumor necrosis factor or “TNF.” Activitiesgenerally ascribed to TNF include: release of other cytokines includingIL-1, IL-6, GM-CSF, and IL-10, induction of chemokines, increase inadhesion molecules, growth of blood vessels, release of tissuedestructive enzymes and activation of T cells. See, for example,Feldmann et al., 1997, Adv. Immunol., 64:283-350, Nawroth et al., 1986,J. Exp. Med., 163:1363-1375; Moser et al., 1989, J. Clin. Invest,83:444-455; Shingu et al., 1993, Clin. Exp. Immunol. 94:145-149; MacNaulet al., 1992, Matrix Suppl., 1:198-199; and Ahmadzadeh et al., 1990,Clin. Exp. Rheumatol. 8:387-391. All of these activities can serve toenhance an inflammatory response.

TNF causes pro-inflammatory actions which result in tissue injury, suchas inducing procoagulant activity on vascular endothelial cells (Pober,et al., J. Immunol. 136:1680 (1986)), increasing the adherence ofneutrophils and lymphocytes (Pober, et al., J. Immunol. 138:3319(1987)), and stimulating the release of platelet activating factor frommacrophages, neutrophils and vascular endothelial cells (Camussi, etal., J. Exp. Med. 166:1390 (1987)). TNF is also associated withinfections (Cerami, et al., Immunol. Today 9:28 (1988)), immunedisorders, neoplastic pathologies (Oliff, et al., Cell 50:555 (1987)),autoimmune pathologies and graft-versus host pathologies (Piguet, etal., J. Exp. Med. 166:1280 (1987)). Among such TNF-associated disordersare congestive heart failure, inflammatory bowel diseases (includingCrohn's disease), arthritis, and asthma.

In particular, TNF plays a central role in gram-negative sepsis andendotoxic shock (Michie, et al., Br. J. Surg. 76:670-671 (1989); Debets,et al., Second Vienna Shock Forum, p. 463-466 (1989); Simpson, et al.,Crit. Care Clin. 5:27-47 (1989); Waage, et al., Lancet 1:355-357 (1987);Hammerle, et al., Second Vienna Shock Forum p. 715-718 (1989); Debets,et al., Crit. Care Med. 17:489-497 (1989); Calandra, et al., J. Infect.Dis. 161:982-987 (1990); Revhaug, et al., Arch. Surg. 123:162-170(1988)), including fever, malaise, anorexia, and cachexia.

TNF initiates its biological effect through its interaction withspecific, cell surface receptors on TNF-responsive cells. There are twodistinct forms of the cell surface tumor necrosis factor receptor(TNFR), designated p75 (or Type 2) and p55 (or Type 1) (Smith et al.,1990, Science 248:1019-1023; Loetscher et al., 1990, Cell 61:351-359).TNFR Type 1 and TNFR Type 2 each bind to both TNFα and TNFβ. Biologicalactivity of TNF is dependent upon binding to either cell surface TNFR.The Type 1 receptor (also termed TNF-R55, TNF-RI, or TNFR-β) is a 55 kdglycoprotein shown to transduce signals resulting in cytotoxic,anti-viral, and proliferative activities of TNF-α. The p75 receptor(also termed TNF-R75, TNFR2, or TNFR-α) is a 75 kDa glycoprotein thathas also been shown to transduce cytotoxic and proliferative signals aswell as signals resulting in the secretion of GM-CSF.

TNF antagonists, such as soluble TNFRs and anti-TNF antibodies, havebeen demonstrated to block TNF activity, causing a decrease in IL-1,GM-CSF, IL-6, IL-8, adhesion molecules and tissue destruction inresponse to TNF (Feldmann et al., 1997). The effect of TNF antagonismutilizing a hamster anti-mouse TNF antibody was tested in a model ofcollagen type II arthritis in DBA/1 mice (Williams et al., 1992, Proc.Natl. Acad. Sci. USA, 89:9784-9788). Treatment initiated after the onsetof disease resulted in improvement in footpad swelling, clinical score,and histopathology of joint destruction. Other studies have obtainedsimilar results using either antibodies (Thorbecke et al., 1992, Proc.Natl. Acad. Sci. USA, 89:7375-7379) or TNFR constructs (Husby et al.,1988, J. Autoimmun. 1:363-71; Tetta et al., 1990, Ann. Rheum. Dis.49:665-667; Wooley et al., 1993, J. Immunol. 151:6602-6607; Piguet etal., 1992, Immunology 77:510-514).

Three specific TNF antagonists are currently FDA-approved: etanercept(Enbrel®), infliximab (Remicade®) and adalimumab (Humira®). One or moreof these drugs is approved for the treatment of rheumatoid arthritis,juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis,ankylosing spondylitis, and inflammatory bowel disease (Crohn's diseaseor ulcerative colitis).

Clinical trials of a recombinant version of the soluble human TNFR (p75)linked to the Fc portion of human IgG1 (sTNFR(p75):Fc, Enbrel®, Immunex)have shown that its administration resulted in significant and rapidreductions in RA disease activity (Moreland et al., 1997, N. Eng. J.Med., 337:141-147). In addition, safety data from a pediatric clinicaltrial for Enbrel® indicates that this drug is generally well-toleratedby patients with juvenile rheumatoid arthritis (JRA) (Garrison et al,1998, Am. College of Rheumatology meeting, Nov. 9, 1998, abstract 584).

As noted above, Enbrel® is a dimeric fusion protein consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)TNFR linked to the Fc portion of human IgG1. The Fc component of Enbrel®contains the CH2 domain, the CH3 domain and hinge region, but not theCH1 domain of IgG1. Enbrel® is produced in a Chinese hamster ovary (CHO)mammalian cell expression system. It consists of 934 amino acids and hasan apparent molecular weight of approximately 150 kilodaltons (Smith etal., 1990, Science 248:1019-1023; Mohler et al., 1993, J. Immunol.151:1548-1561; U.S. Pat. No. 5,395,760 (Immunex Corporation, Seattle,Wash.); U.S. Pat. No. 5,605,690 (Immunex Corporation, Seattle, Wash.).

Enbrel® is currently indicated for reduction in signs and symptoms ofmoderately to severely active rheumatoid arthritis in patients who havehad an inadequate response to one or more disease-modifyingantirheumatic drugs (DMARDs). Enbrel® can be used in combination withmethotrexate in patients who do not respond adequately to methotrexatealone. Enbrel® is also indicated for reduction in signs and symptoms ofmoderately to severely active polyarticular-course juvenile rheumatoidarthritis in patients who have had an inadequate response to one or moreDMARDs (May 28, 1999). Enbrel® is given to RA patients at 25 mg twiceweekly as a subcutaneous injection.

Currently, treatments using ENBREL preparations are administeredsubcutaneously twice weekly, which is costly, unpleasant andinconvenient for the patient. Accordingly, the present disclosureprovides TNF antagonists comprising a soluble (e.g., extracellular)portion of an TNF binding receptor (e.g., Enbrel®) that has beenmodified to introduce at least one non-endogenous N-linked glycosylationsite to increase the serum half-life of the modified polypeptiderelative to the serum half-life of the soluble TNF receptor lacking theintroduced glycosylation site.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

The term “pharmacokinetic properties” refers to the absorption,distribution, metabolism and excretion of a bioactive agent (e.g., smallmolecule, polypeptide drug, etc.).

2. Glycovariant Fc Fusion Proteins

Provided herein are glycovariant fusion proteins having an Fc domainfrom an immunoglobulin molecule linked to a heterologous polypeptide.The glycovariant fusion proteins contain at least one non-endogenousN-linked glycosylation site outside of the Fc domain of the fusionprotein. The Fc domain may be linked either directly, or indirectly viaa polypeptide linker, to the heterologous polypeptide. A non-endogenousglycosylation site may be introduced into the heterologous polypeptide,the linker, or both.

A “non-endogenous”, “introduced”, or “novel” glycosylation site refersto a glycosylation site that is not present in an unmodified version ofthe polypeptide. Accordingly, the glycovariant fusion proteins describedherein have at least one additional N-linked glycosylation site ascompared to the number of glycosylation sites present in the unmodifiedversion of the fusion protein. For example, if the unmodified version ofthe fusion protein has two glycosylation sites outside of the Fc domainand one N-linked glycosylation site is introduced into the heterologouspolypeptide, then the glycovariant fusion protein will have threeN-linked glycosylation sites including two native glycosylation sitesand one introduced glycosylation site. It should be understood thatmodification of a fusion protein to increase the number of glycosylationsites is not limited to a particular “wild-type” amino acid sequencebecause naturally occurring or man-made variants can also be modifiedaccording to the methods of this invention to increase the number ofglycosylation sites.

It is well known that some proteins can support a large number of sugarside chains. The distance between O-linked glycosylation sites can be asfew as every other amino acid (see, e.g., Kolset & Tveit (2008) Cell.MoI. Life Sci 65: 1073-1085 and Kiani et al. (2002) Cell Research 12(1):19-32). For N-linked glycosylation sites, the distance between sites canbe as few as three, four, five or six amino acids (see, e.g., Lundin etal. (2007) FEBS Letters 581:5601-5604 (2007); Apweiler et al. (1991)Biochimica et Biophysica Acta 1473:4-8, the entire contents of each ofwhich are incorporated by reference herein). Accordingly, in certainembodiments at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or moreN-linked glycosylation sites can be added to a fusion protein describedherein. In certain embodiments, a glycovariant fusion protein of thedisclosure comprises at least one glycosylated amino acid per each 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, or 200 amino acids. In certain embodiments, each N-linkedglycosylated amino acid is separated by at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more aminoacids from any other N-linked, O-linked, or N-linked and O-linkedglycosylated amino acid.

As used herein, a “glycosylation site” can mean a sugar attachmentconsensus sequence (i.e., a series of amino acids that act as aconsensus sequence for attaching a sugar, either mono-, oligo-, orpolysaccharides to an amino acid sequence) or it can mean the actualamino acid residue to which the sugar moiety is covalently linked. Thesugar moiety can be a monosaccharide (simple sugar molecule), anoligosaccharide, or a polysaccharide.

N-linked glycosylation sites may be introduced into a fusion proteineither by modifying the amino acid sequence of the protein or bychemically modifying an amino acid residue in the fusion protein to adda sugar moiety. In preferred embodiments, the fusion proteins describedherein are modified to introduce (e.g., by insertion, deletion, orsubstitution of specified amino acids) at least one N-linkedglycosylation site having the consensus sequence NXT/S(asparagine-X-serine/threonine), wherein X is any amino acid other thanproline. Another means of increasing the number of carbohydrate moietieson a protein is by chemical or enzymatic coupling of glycosides to thepolypeptide. For example, depending on the coupling mode used, a sugarmoiety may be attached to (a) arginine and histidine; (b) free carboxylgroups; (c) free sulfhydryl groups such as those of cysteine; (d) freehydroxyl groups such as those of serine, threonine, or hydroxyproline;(e) aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (f) the amide group of glutamine. See e.g., WO 87/05330and Aplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306,incorporated by reference herein. In exemplary embodiments, a sugarmoiety is coupled to an arginine residue to produce an N-linked glycan.

In exemplary embodiments, sugar moieties are added to an introducedglycosylation site using the cellular machinery by expressing the fusionprotein in a host cell. Generally, fusion proteins will be expressed ina mammalian cell line that provides proper glycosylation, such as HEK293or CHO cell lines, or other mammalian expression cell lines.

In certain embodiments, sugar moieties may be added to an introducedglycosylation site using a non-mammalian host cell, such as a yeast,bacteria or insect cells, that has been engineered to producemammalian-like glycosylation. Cell lines having genetically modifiedglycosylation pathways have been developed that carry out a sequence ofenzyme reactions mimicking the processing of glycoproteins in humans.Recombinant proteins expressed in these engineered cells yieldglycoproteins similar, if not substantially identical, to their humancounterparts (e.g., mammalian-like glycosylation).

Techniques for genetically modifying host cells to alter theglycosylation profile of expressed peptides are well-known. See, e.g.,Altmann et al. (1999, Glycoconjugate J. 16: 109-123), Ailor et al.(2000, Glycobiology 10(8): 837-847), Jarvis et al., (In vitrogenConference, March, 1999, abstract), Hollister and Jarvis, (2001,Glycobiology 11(1): 1-9), and Palacpac et al., (1999, PNAS USA 96:4697), Jarvis et al., (1998. Curr. Opin. Biotechnol. 9:528-533),Gemgross (U.S. Patent Publication No. 20020137134), all of whichdisclose techniques to “humanize” insect or plant cell expressionsystems by transfecting insect or plant cells with glycosyltransferasegenes.

Techniques also exist to genetically alter the glycosylation profile ofpeptides expressed in prokaryotic systems. E. coli has been engineeredwith various glycosyltransferases from the bacteria Neisseriameningitidis and Azorhizobium to produce oligosaccharides in vivo(Bettler et al., 1999, Glycoconj. J. 16:205-212). E. coli which has beengenetically engineered to over-express Neisseria meningitidis β1,3 Nacetyl glucosaminyltransferase lgta gene will efficiently glycosylateexogenous lactose (Priem et al., 2002, Glycobiology 12:235-240).

Fungal cells have also been genetically modified to produce exogenousglycosyltransferases (Yoshida et al., 1999, Glycobiology, 9(1):53-58;Kalsner et al., 1995, Glycoconj. J. 12:360-370; Schwientek and Ernst,1994, Gene 145(2):299-303; Chiba et al, 1995, Biochem J. 308:405-409).

In certain embodiments, host cells expressing an Fc fusion protein ofthe disclosure may be a eukaryotic or prokaryotic cell expressing one ormore exogenous glycosyltransferase enzymes and/or one or more exogenousglycosidase enzymes, wherein expression of a recombinant glycopeptide inthe host cell results in the production of a recombinant glycopeptidehaving a “human” glycan structures.

In some embodiments the heterologous glycosyltransferase enzyme usefulin the cell may be selected from a group consisting of any knownglycosyltransferase enzyme included for example, in the list ofGlycosyltransferase Families available in Taniguchi et al. (2002,Handbook of Glycosyltransferases and Related Genes, Springer, N.Y.).

In some embodiments, the host cell may be a eukaryotic or prokaryoticcell wherein one or more endogenous glycosyltransferase enzymes and/orone or more endogenous glycosidase enzymes have been inactivated suchthat expression of a recombinant glycopeptide in the host cell resultsin the production of a recombinant glycopeptide having a “human” glycanstructure.

In some embodiments, the host cell may express heterologousglycosyltransferase enzymes and/or glycosidase enzymes while at the sametime one or more endogenous glycosyltransferase enzymes and/orglycosidase enzymes are inactivated. Endogenous glycosyltransferaseenzymes and/or glycosidase enzymes may be inactivated using anytechnique known to those skilled in the art including, but not limitedto, antisense techniques and techniques involving insertion of nucleicacids into the genome of the host cell.

In exemplary embodiments, the glycovariant fusion proteins describedherein have increased stability and/or an increased serum half liferelative to the unmodified form of the fusion protein. In exemplaryembodiments, the serum half life of the glycovariant fusion protein isincreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 250% or 300% or morerelative to the unmodified form of the fusion protein. In certainembodiments, serum half-life of a modified Fc fusion protein and afusion polypeptide lacking the additional glycosylation site aremeasured in the same animal model or species for comparison. Inexemplary embodiments, the serum half-life is measured in apharmacokinetic rat assay or a pharmacokinetic monkey assay as describedherein (see e.g., Examples 4 and 5).

In exemplary embodiments, introduction of the one or more non-endogenousglycosylation sites does not significantly affect one or more biologicalactivities of the heterologous polypeptide portion of the fusionprotein. For example, one or more biological activities of theheterologous polypeptide portion of the fusion protein may be affectedby less than 10-fold, 5-fold, 3-fold, 2.5-fold, 2-fold, 1.5-fold,1-fold, 0.5-fold or less. Examples of biological activities of theheterologous polypeptide portion are described further herein andinclude for example, protein-protein interaction, ligand binding,physiological activity, etc.

In exemplary embodiments, one or more non-endogenous glycosylation sitesare introduced into the heterologous portion of an Fc fusion protein.The heterologous portion of the Fc fusion protein may comprise acatalytic domain or a ligand binding domain. As used herein, a “ligandbinding domain” refers to a region on a protein that interacts withanother molecule, e.g., a ligand. For example, a ligand binding domainmay refer to a region on a protein that is bound by an antibody, aregion on an antibody that binds to an antigen, a region on a receptorthat binds to a ligand, a region on a ligand that binds to a receptor, aregion on a first polypeptide that binds to a second polypeptide, aregion on a polypeptide that binds to a small molecule, etc. Inexemplary embodiments, a ligand binding domain is a region in theextracellular domain of a transmembrane receptor protein that binds to aligand. As used herein a “catalytic domain” refers to a region having afunctional activity. Examples of catalytic domains include, for example,kinase domains, phosphatase domains, protease domains, etc.

In exemplary embodiments, one or more non-endogenous glycosylation sitesare introduced into a surface exposed region that is flexible orunstructured, e.g., loop regions that connect α-helices and/or β-sheets.In general, a protein is a polypeptide chain having secondary andtertiary structure. The secondary structure of a polypeptide involvesfolding of a polypeptide chain into two common structural domains calledα-helices and β-sheets. The tertiary structure of a protein consists ofcombinations of secondary structures, α-helices and β-sheets, connectedby loop regions of various lengths and irregular shape. Generally,combinations of secondary structure elements form a stable hydrophobiccore, while the loop regions are presented at the surface of theprotein. As loop regions are solvent exposed, they are generally moresusceptible to nucleophilic attack/peptide bond cleavage by variousreactive molecules and protein enzymes (e.g., proteases) than theinternal, structurally stable secondary structural domains (e.g.,α-helix and β-sheet domains).

In certain embodiments, it may be desirable to remove one or morenaturally occurring glycosylation sites (e.g., glycosylation sites inthe unmodified form of the protein) in the heterologous polypeptideportion of the fusion protein. For example, in order to change thespacing of the N-linked glycosylation sites, it may be desirable toremove a naturally occurring glycosylation site and then introduce twonon-endogenous glycosylation sites at desired locations. In addition, itmay be desirable to remove one or more of the naturally occurringO-linked glycosylation sites occurring in the fusion protein.

N-linked and O-linked glycosylation sites may be removed by eliminatinga consensus amino acid sequence or by chemical or enzymatic cleavage ofthe sugar moiety from the amino acid residue. For example, N-linkedglycosylation sites may be removed by amino acid substitutions ordeletions at one or both of the first or third amino acid positions ofan N-linked glycosylation recognition site (i.e. NXT/S), and/or aminoacid deletion at the second position of the tripeptide sequence.O-linked glycosylation sites may be removed by the addition, deletion,or substitution of one or more serine or threonine residues and/or thedisruption of an O-linked consensus sequence as set forth below.Alternatively, removal of one or more naturally occurring carbohydratemoieties present on a polypeptide may be accomplished chemically and/orenzymatically. Chemical deglycosylation may involve, for example,exposure of a glycosylated polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on glycosylated polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.(1987) Meth. Enzymol. 138:350.

In certain embodiments, it may be desirable to introduce one or moreO-linked glycosylation sites in addition to the one or morenon-endogenous N-linked glycosylation sites. O-linked glycosylationsites may be added to protein by introducing an O-linked glycosylationconsensus sequence into the polypeptide. Exemplary O-linked consensussequences include, for example, CXXGGT/S-C (SEQ ID NO:19), NSTE/DA (SEQID NO:20), NITQS (SEQ ID NO:21), QS1QS (SEQ ID NO:22), 0/E-FI1RZK-V (SEQID NO:23), C-E/D-SN, and GGSC-K/R (SEQ ID NO:24). Alternatively,O-linked sugar moieties may be introduced by chemically modifying anamino acid in the polypeptide. See e.g., WO 87/05330 and Aplin andWriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, incorporated byreference herein.

In certain embodiments, the glycovariant polypeptides described hereinmay comprise additional domains, such as leader sequences, linkers,and/or purification/identification tags.

In certain embodiments, serum half-life of hyperglycosylated proteinsdescribed herein may be improved by the deletion of C- and/or N-terminalresidues of a protein domain, such as the extracellular domain of areceptor (e.g., TNFR2 or CTLA4). Preferably, such deletions are designedso as not to disrupt any secondary structure elements, such asalpha-helices or beta-sheets. Deletions may be 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 21, 25, 30 or more amino acids at the N- and/orC-terminus.

In some embodiments, fusion proteins may comprise a signal sequence, forexample, a honey bee mellitin leader (HBML): MKFLVNVALVFMVVYISYIYA (SEQID NO: 10); Tissue Plasminogen Activator (TPA) leader:MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 11); or (iii) a native leadersequence.

Fusion proteins of the disclosure may optionally comprise a linkerdomain. In certain embodiments, fusion proteins comprise a relativelyunstructured linker positioned between the Fc domain and theheterologous polypeptide domain. This unstructured linker may be anartificial sequence of 1, 2, 3, 4, or 5 amino acids, or a length between5 and 10, 15, 20, 30, 50 or more amino acids, that are relatively freeof secondary structure. A linker may be rich in glycine and prolineresidues and may, for example, contain a single sequence ofthreonine/serine and glycines or repeating sequences of threonine/serineand glycines (e.g., TG₃ (SEQ ID NO: 12) or SG₃ (SEQ ID NO: 13) singletsor repeats). In certain embodiments, one or more of the introducedglycosylation sites can be located in the linker domain. In certainembodiments one or more of the introduced glycosylation sites can belocated at a position other than the linker domain or outside of theregion that is within ten amino acids of a junction between polypeptidesthat creates a junction sequence that is not a naturally occurringsequence. In certain embodiments, a hyperglycosylated protein does notinclude multiple repeats of, for example, a extracellular domain of areceptor. Thus, for example, a TNFR2-Fc hyperglycosylated polypeptidedescribed herein may preferably have a single TNFR2 ECD portion fused toan Fc (which then dimerize to make a complete Fc fusion protein), ratherthan having two or more TNFR2 ECD portions fused in tandem and thenfused to an Fc.

Fusion proteins of the disclosure may include a domain that facilitatesisolation and/or detection of the fusion protein. Well known examples ofsuch domains include, but are not limited to, polyhistidine, Glu-Glu,glutathione S transferase (GST), thioredoxin, protein A, protein G,maltose binding protein (MBP), or human serum albumin. A fusion domainmay be selected so as to confer a desired property. For example, somefusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QIAexpress™ system (Qiagen)useful with (HIS₆) (SEQ ID NO:25) fusion partners. As another example, afusion domain may be selected so as to facilitate detection of thepolypeptides. Examples of such detection domains include the variousfluorescent proteins (e.g., GFP) as well as “epitope tags,” which areusually short peptide sequences for which a specific antibody isavailable. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushemagglutinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an Fc domain may be placed C-terminal to aheterologous polypeptide domain, or, alternatively, a heterologouspolypeptide domain may be placed C-terminal to an Fc domain. The Fcdomain and the heterologous domain need not be adjacent in a fusionprotein, and additional domains or amino acid sequences may be includedC- or N-terminal to either domain or between the domains.

In certain embodiments, the glycovariant fusion proteins may include oneor more additional modified amino acid residues selected from: aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent.As a result of such post-translational modifications, the fusionproteins described herein may contain non-amino acid elements, such aspolyethylene glycols, lipids, and phosphates. Different cells (such asCHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK293) have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the polypeptides of the invention.

Fusion proteins of the disclosure are generally of higher molecularweight, being at least 30, 40, 50, 60, 70, 80, 90, 100, or 110 kDa, ormore, in size.

In exemplary embodiments, the disclosure provides TNFR2-Fc glycovariantfusion proteins having at least one non-endogenous N-linkedglycosylation sites outside of the Fc domain. In certain embodiments, aglycovariant fusion protein of the invention may comprise an amino acidsequence that is at least 75% identical to an amino acid sequenceselected from SEQ ID NOs: 6, 7, 8, 46, 47, 53 or 54. In certain cases,the glycovariant fusion protein comprises an amino acid sequence atleast 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from SEQ ID NOs: 6, 7, 8, 46, 47, 53 or 54.

In exemplary embodiments, the disclosure provides CTLA4-Fc glycovariantfusion proteins having at least one non-endogenous N-linkedglycosylation sites outside of the Fc domain. In certain embodiments, aglycovariant fusion protein of the invention may comprise an amino acidsequence that is at least 75% identical to an amino acid sequenceselected from SEQ ID NOs: 50 or 52. In certain cases, the glycovariantfusion protein has an amino acid sequence at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to an amino acid sequence selected fromSEQ ID NOs: 50 or 52.

In certain embodiments, the glycovariant Fc fusion proteins describedherein do not contain an extracellular domain of an ActRIIb receptorand/or a variable region of an antibody.

Fc Domains

The glycovariant fusion proteins described herein comprise animmunoglobulin heavy chain Fc domain fused to a heterologouspolypeptide. Fusions with the Fc portion of an immunoglobulin are knownto confer desirable pharmacokinetic properties on a wide range ofproteins. The term “Fc region” or “Fc domain” as used herein refers to aC-terminal region of an immunoglobulin heavy chain, including nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminus ofthe immunoglobulin. The C-terminal lysine (residue 447 according to theEU numbering system) of the Fc region may be removed, for example, whenrecombinantly engineering the nucleic acid encoding a heavy chain of theantibody. Unless indicated otherwise, herein the numbering of theresidues in an immunoglobulin heavy chain is that of the EU index as inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991), expressly incorporated herein by reference. The “EU index as inKabat” refers to the residue numbering of the human IgG1 EU antibody.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

In preferred embodiments, the Fc fusion protein described herein have aheterologous portion fused to the N-terminus of the C-terminal portionof an immunoglobulin Fc domain. Preferably, a sequence beginning in thehinge region just upstream of the papain cleavage site, e.g. taking thefirst residue of heavy chain constant region to be 114 or analogoussites of other immunoglobulins, is used in the fusion. In oneembodiment, the heterologous domain is fused to the hinge region and CH2and CH3 or CH1, hinge, CH2 and CH3 domains of an IgG1, IgG2, IgG3 orIgG4 heavy chain. Optionally, as shown in SEQ ID NO: 54 and in otherexamples here, the fusion protein may use a truncated hinge-CH2-CH3portion of an IgG1, IgG2, IgG3 or IgG4 heavy chain, such that theprotein does not contain all of the domains running from the hingeportion through the CH3 domain. In some embodiments, the precise site atwhich the fusion is made is not critical, and the optimal site can bedetermined by routine experimentation.

For human Fc domains, the use of human IgG1 and IgG3 immunoglobulinsequences is preferred. A major advantage of using IgG1 is that an IgG1fusion protein can be purified efficiently on immobilized protein A. Incontrast, purification of IgG3 fusion proteins requires protein G, asignificantly less versatile medium. However, other structural andfunctional properties of immunoglobulins should be considered whenchoosing the Ig fusion partner for a particular construction. Forexample, the IgG3 hinge is longer and more flexible, so it canaccommodate a larger heterologous portion that may not fold or functionproperly when fused to IgG1. Another consideration may be valency; IgGimmunoadhesins are bivalent homodimers, whereas Ig subtypes like IgA andIgM may give rise to dimeric or pentameric structures, respectively, ofthe basic Ig homodimer unit.

For Fc fusion proteins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG1, IgG2 and IgG4 all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG4 does not activate complement,and IgG2 is significantly weaker at complement activation than IgG1.Moreover, unlike IgG1, IgG2 does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG3 is optimal for complement activation,its in vivo half-life in approximately one third of the other IgGisotypes.

Another important consideration for Fc fusion proteins designed to beused as human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-defined allotypes are preferred. For example, IgG1 hasonly four serologically-defined allotypic sites, two of which (GIm and2) are located in the Fc region; and one of these sites Glml, isnon-immunogenic. In contrast, there are 12 sero logically-definedallotypes in IgG3, all of which are in the Fc region; only three ofthese sites (G3m5, 11 and 21) have one allotype which isnon-immunogenic. Thus, the potential immunogenicity of an IgG3 fusionprotein is greater than that of an IgG1 fusion protein.

In certain embodiments, an Fc domain used in the Fc fusion proteins maycomprise one or more alterations as compared to the wild-type Fc domain.These Fc domains would nonetheless retain substantially the samecharacteristics required for therapeutic utility as compared to theirwild-type counterpart. For example, it is thought that certainalterations can be made in the Fc region that would result in altered(i.e., either improved or diminished) CIq binding and/or ComplementDependent Cytotoxicity (CDC), e.g., as described in WO99/51642. See alsoDuncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S.Pat. No. 5,624,821; and WO94/29351 concerning other examples of Fcregion variants. WO00/42072 (Presta) and WO 2004/056312 (Lowman)describe antibody variants with improved or diminished binding to FcRs.The content of these patent publications are specifically incorporatedherein by reference. See, also, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies with increased half-lives and improvedbinding to the neonatal Fc receptor (FcRn), which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are describedin US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc regon with one or more substitutions therein which improve binding of theFc region to FcRn. Polypeptide variants with altered Fc region aminoacid sequences and increased or decreased CIq binding capability aredescribed in U.S. Pat. No. 6,194,551 and the International PublicationWO99/51642. See, also, Idusogie et al. J. Immunol. 164: 4178-4184(2000). The contents of these are specifically incorporated herein byreference.

An exemplary Fc domain, which uses a truncated hinge portion is shownbelow.

(e.g., SEQ ID NO: 14) THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

An example of an advantageous linker (TG₃) (SEQ ID NO:12) and Fc domaincombined is shown below:

(SEQ ID NO: 18) TGGG THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wild-typeFc domain. In other cases, the mutant Fc domain having one or more ofthese mutations (e.g., Asn-434 mutation) has increased ability ofbinding to the MHC class I-related Fc-receptor (FcRN) relative to awild-type Fc domain.

Heterologous Polypeptides

The glycovariant fusion proteins described herein also comprise aheterologous polypeptide portion fused either directly or indirectly tothe Fc domain. The heterologous polypeptide portion may be anypolypeptide. In exemplary embodiments the heterologous polypeptideportion has a molecular weight of at least 10, 15, 20, 25, 30, 35, 40,50, 60, 70, 80, 90 or 100 kDa or greater.

Heterologous polypeptide portions may be a therapeutic protein, orfragments thereof, such as growth factors, enzymes, serum enzymes,endocrine factors such as GLP1, bone morphogenetic proteins and solublereceptor fragments. Exemplary heterologous polypeptides include growthfactors, such as hepatocyte growth factor (HGF), nerve growth factors(NGF), epidermal growth factors (EGF), Cytotoxic T-Lymphocyte Antigen 4(CTLA4), fibroblast growth factors (FGF), transforming growth factors(e.g., TGF-alpha, TGF-beta, TGF-beta2, TGF-beta3), vascular endothelialgrowth factors (VEGF; e.g., VEGF-2), interferons (e.g., INF-alpha,INF-beta) and insulin. Other exemplary heterologous polypeptides includeenzymes, such as alpha-galactosidase (e.g., Fabrazyme™). Other exemplaryheterologous polypeptides include bone morphogenetic proteins (BMP),erythropoietines (EPO), myostatin, and tumor necrosis factors (e.g.,TNF-α). Other exemplary heterologous polypeptides include extracellulardomains of transmembrane receptors, including any naturally occurringextracellular domain of a cellular receptor as well as any variantsthereof (including mutants, fragments and peptidomimetic forms).

In exemplary embodiments, the heterologous polypeptide portion is anextracellular domain of a receptor from the TNF/NGF family of receptorsor the CTLA4 receptor. Examples of soluble receptor polypeptidesinclude, for example, SEQ ID NOs: 2, 5, 6, 7, 8, 16, 44, 46, 47, 50, 52,53 or 54. In preferred embodiments, an extracellular receptor domainincluded in the glycovariant fusion proteins having at least onenon-endogenous N-linked glycosylation site retains the ability to bind aligand of the naturally occurring receptor. In certain embodiments, anon-endogenous N-linked glycosylation site is introduced outside of theligand binding pocket of the extracellular receptor domain. Preferably,glycosylation of the introduced glycosylation site does notsignificantly affect the binding of the receptor protein to the solubleligand. In exemplary embodiments, glycosylation of the introducedglycosylation sited does not reduce binding of the extracellularreceptor domain to its ligand by more than 10-fold, 5-fold, 3-fold,2.5-fold, 2-fold, 1.5-fold, 1-fold, 0.5-fold, or less. Preferably, asoluble receptor domain comprising at least one non-endogenous N-linkedglycosylation site will bind to a specified ligand with a dissociationconstant of less than 1 μM or less than 100, 10, or 1 nM.

In one embodiment, the glycovariant fusion proteins described hereincomprises a heterologous polypeptide portion that comprises anextracellular domain of a receptor from the nerve growth factor(NGF)/tumor necrosis factor (TNF) receptor family. See e.g., M. Lotz, etal., J. Leukocyte Biology, 60:1-7 (1990). The NGF/TNF receptor familyincludes, for example, nerve growth factor receptor (NGFR), TNFR1 (orTNFR55), TNFR2 (or TNFR75), the TNF receptor-related protein (TNFRrp),CD40, the Hodgkin's antigen CD30, the T cell antigen CD27, Fas/APO-1,OX-40, and 4-1BB/ILA. In exemplary embodiments, the fusion proteinsdescribed herein comprise an extracellular domain of TNFR2 having atleast one non-endogenous N-linked glycosylation site.

As used herein, the term “TNFR2” refers to a family of tumor necrosisfactor receptor type 2 (TNFR2) proteins from any species and variantsderived from such TNFR2 proteins by mutagenesis or other modification.Members of the TNFR2 family are generally transmembrane proteins,composed of a ligand-binding extracellular domain with a cysteine-richregion, a transmembrane domain, and a cytoplasmic domain with predictedintracellular signaling activity.

The term “TNFR2 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of a TNFR2 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. For example, TNFR2polypeptides include polypeptides derived from the sequence of any knownTNFR2 having a sequence at least about 80% identical to the sequence ofan TNFR2 polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% orgreater identity. For example, a TNFR2 polypeptide of the invention maybind to TNFα or TNFβ and inhibit the function of a TNFR2 protein and/orTNFα or TNFβ. Examples of TNFR2 polypeptides include human TNFR2precursor polypeptide (SEQ ID NO:1) and soluble human TNFR2 polypeptides(e.g., SEQ ID NOs: 2, 5, 6, 7, 8, 16, 44, 46, 47, 53 or 54).

The human TNFR2 precursor protein sequence is as follows:

(SEQ ID NO: 1) MAPVAVWAAL AVGLELWAAA HA LPAQVAFT PYAPEPGSTCRLREYYDQTA QMCCSKCSPG QHAKVFCTKT SDTVCDSCEDSTYTQLWNWV PECLSCGSRC SSDQVETQAC TREQNRICTCRPGWYCALSK QEGCRLCAPL RKCRPGFGVA RPGTETSDVV CKPCAPGTFS  NTTSSTDICR PHQICNVVAI PG NAS MDAVCTSTSPTRSMA PGAVHLPQPV STRSQHTQPT PEPSTAPSTSFLLPMGPSPP AEGSTGDEPK SCDKTHTCPP CPAPELLGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEMTKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK*

The signal peptide is single underlined; the extracellular domain is inbold and the potential N-linked glycosylation sites are doubleunderlined.

The human TNFR2 soluble (extracellular), processed polypeptide sequenceis as follows:

(SEQ ID NO: 2) LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQHAKVFCTKTSD TVCDSCEDST YTQLWNWVPE CLSCGSRCSSDQVETQACTR EQNRICTCRP GWYCALSKQE GCRLCAPLRKCRPGFGVARP GTETSDVVCK PCAPGTFS

  T SSTDICRPH QICNVVAIPG 

MDAVCTS TSPTRSMAPG AVHLPQPVST RSQHTQPTPE PSTAPSTSFL LPMGPSPPAE GSTGD

Naturally occurring N-linked glycosylation sites are underlined. Asreported herein, the amino terminal leucine may be eliminated, leaving aproline residue at the amino-terminus.

As described in the examples, a truncation of up to 21 amino acids atthe C-terminal end of TNFR2 extracellular domain provided a highlyactive protein. The sequence of the TNFR2 CΔ21, processed polypeptide isas follows:

(SEQ ID NO: 44) LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQHAKVFCTKTSD TVCDSCEDST YTQLWNWVPE CLSCGSRCSSDQVETQACTR EQNRICTCRP GWYCALSKQE GCRLCAPLRKCRPGFGVARP GTETSDVVCK PCAPGTFS

  T SSTDICRPH QICNVVAIPG 

MDAVCTS TSPTRSMAPG AVHLPQPVST RSQHTQPTPE PSTA

Naturally occurring N-linked glycosylation sites are underlined. Asreported herein, the amino terminal leucine may be eliminated, leaving aproline residue at the amino-terminus.

Naturally occurring N-linked glycosylation sites are underlined. Asreported herein, the amino terminal leucine may be eliminated, leaving aproline residue at the amino-terminus.

The nucleic acid sequence encoding human TRFR2 precursor protein is asfollows (Genbank entry NM_(—)001066.2):

(SEQ ID NO: 3) ATGGCGCCCG TCGCCGTCTG GGCCGCGCTG GCCGTCGGACTGGAGCTCTG GGCTGCGGCG CACGCCTTGC CCGCCCAGGTGGCATTTACA CCCTACGCCC CGGAGCCCGG GAGCACATGCCGGCTCAGAG AATACTATGA CCAGACAGCT CAGATGTGCTGCAGCAAATG CTCGCCGGGC CAACATGCAA AAGTCTTCTGTACCAAGACC TCGGACACCG TGTGTGACTC CTGTGAGGACAGCACATACA CCCAGCTCTG GAACTGGGTT CCCGAGTGCTTGAGCTGTGG CTCCCGCTGT AGCTCTGACC AGGTGGAAACTCAAGCCTGC ACTCGGGAAC AGAACCGCAT CTGCACCTGCAGGCCCGGCT GGTACTGCGC GCTGAGCAAG CAGGAGGGGTGCCGGCTGTG CGCGCCGCTG CGCAAGTGCC GCCCGGGCTTCGGCGTGGCC AGACCAGGAA CTGAAACATC AGACGTGGTGTGCAAGCCCT GTGCCCCGGG GACGTTCTCC AACACGACTTCATCCACGGA TATTTGCAGG CCCCACCAGA TCTGTAACGTGGTGGCCATC CCTGGGAATG CAAGCATGGA TGCAGTCTGCACGTCCACGT CCCCCACCCG GAGTATGGCC CCAGGGGCAGTACACTTACC CCAGCCAGTG TCCACACGAT CCCAACACACGCAGCCAACT CCAGAACCCA GCACTGCTCC AAGCACCTCCTTCCTGCTCC CAATGGGCCC CAGCCCCCCA GCTGAAGGGAGCACTGGCGA CTTCGCTCTT CCAGTTGGAC TGATTGTGGGTGTGACAGCC TTGGGTCTAC TAATAATAGG AGTGGTGAACTGTGTCATCA TGACCCAGGT GAAAAAGAAG CCCTTGTGCCTGCAGAGAGA AGCCAAGGTG CCTCACTTGC CTGCCGATAAGGCCCGGGGT ACACAGGGCC CCGAGCAGCA GCACCTGCTGATCACAGCGC CGAGCTCCAG CAGCAGCTCC CTGGAGAGCTCGGCCAGTGC GTTGGACAGA AGGGCGCCCA CTCGGAACCAGCCACAGGCA CCAGGCGTGG AGGCCAGTGG GGCCGGGGAGGCCCGGGCCA GCACCGGGAG CTCAGATTCT TCCCCTGGTGGCCATGGGAC CCAGGTCAAT GTCACCTGCA TCGTGAACGTCTGTAGCAGC TCTGACCACA GCTCACAGTG CTCCTCCCAAGCCAGCTCCA CAATGGGAGA CACAGATTCC AGCCCCTCGGAGTCCCCGAA GGACGAGCAG GTCCCCTTCT CCAAGGAGGAATGTGCCTTT CGGTCACAGC TGGAGACGCC AGAGACCCTGCTGGGGAGCA CCGAAGAGAA GCCCCTGCCC CTTGGAGTGCCTGATGCTGG GATGAAGCCC AGTTAA.

The nucleic acid sequence encoding a human TNFR2 soluble (extracellular)polypeptide is as follows:

(SEQ ID NO: 4) TTGC CCGCCCAGGT GGCATTTACA CCCTACGCCC CGGAGCCCGGGAGCACATGC CGGCTCAGAG AATACTATGA CCAGACAGCTCAGATGTGCT GCAGCAAATG CTCGCCGGGC CAACATGCAAAAGTCTTCTG TACCAAGACC TCGGACACCG TGTGTGACTCCTGTGAGGAC AGCACATACA CCCAGCTCTG GAACTGGGTTCCCGAGTGCT TGAGCTGTGG CTCCCGCTGT AGCTCTGACCAGGTGGAAAC TCAAGCCTGC ACTCGGGAAC AGAACCGCATCTGCACCTGC AGGCCCGGCT GGTACTGCGC GCTGAGCAAGCAGGAGGGGT GCCGGCTGTG CGCGCCGCTG CGCAAGTGCCGCCCGGGCTT CGGCGTGGCC AGACCAGGAA CTGAAACATCAGACGTGGTG TGCAAGCCCT GTGCCCCGGG GACGTTCTCCAACACGACTT CATCCACGGA TATTTGCAGG CCCCACCAGATCTGTAACGT GGTGGCCATC CCTGGGAATG CAAGCATGGATGCAGTCTGC ACGTCCACGT CCCCCACCCG GAGTATGGCCCCAGGGGCAG TACACTTACC CCAGCCAGTG TCCACACGATCCCAACACAC GCAGCCAACT CCAGAACCCA GCACTGCTCCAAGCACCTCC TTCCTGCTCC CAATGGGCCC CAGCCCCCCA GCTGAAGGGA GCACTGGCGA C.

The nucleic acid sequence encoding a human TNFR2 soluble (extracellular)polypeptide with a 21 amino acid deletion at the N-terminus is asfollows:

(SEQ ID NO: 45) TTGC CCGCCCAGGT GGCATTTACA CCCTACGCCC CGGAGCCCGGGAGCACATGC CGGCTCAGAG AATACTATGA CCAGACAGCTCAGATGTGCT GCAGCAAATG CTCGCCGGGC CAACATGCAAAAGTCTTCTG TACCAAGACC TCGGACACCG TGTGTGACTCCTGTGAGGAC AGCACATACA CCCAGCTCTG GAACTGGGTTCCCGAGTGCT TGAGCTGTGG CTCCCGCTGT AGCTCTGACCAGGTGGAAAC TCAAGCCTGC ACTCGGGAAC AGAACCGCATCTGCACCTGC AGGCCCGGCT GGTACTGCGC GCTGAGCAAGCAGGAGGGGT GCCGGCTGTG CGCGCCGCTG CGCAAGTGCCGCCCGGGCTT CGGCGTGGCC AGACCAGGAA CTGAAACATCAGACGTGGTG TGCAAGCCCT GTGCCCCGGG GACGTTCTCCAACACGACTT CATCCACGGA TATTTGCAGG CCCCACCAGATCTGTAACGT GGTGGCCATC CCTGGGAATG CAAGCATGGATGCAGTCTGC ACGTCCACGT CCCCCACCCG GAGTATGGCCCCAGGGGCAG TACACTTACC CCAGCCAGTG TCCACACGATCCCAACACAC GCAGCCAACT CCAGAACCCA GCACTGCT.

The disclosure demonstrates that the addition of one or more N-linkedglycosylation sites (NXS/T) increases the serum half-life of a TNFR2-Fcfusion protein relative an unmodified TNFR2-Fc protein. N-linkedglycosylation sites may be introduced with minimal effort by introducingan N in the correct position with respect to a pre-existing S or T, orby introducing an S or T at a correct position with respect to apre-existing N. Particularly suitable sites for the introduction ofnon-endogenous NXS/T sequences in TNFR2 include, for example, aminoacids 25-27 (DQT), 26-28 (QIA), 133-135 (ETS), and 231-233 (GST) inrelation to SEQ ID NO: 2. For example, desirable alterations that wouldintroduce an N-linked glycosylation site are: D25N, Q26N, A28S, A28T,E133N, and G231N. In some instances, several amino acids may be modifiedto introduce a non-endogenous NXS/T sequence. For example, a doublesubstitution of Q26N/A28S (or A28T) provides both the N and S (or T)residues necessary to introduce a novel N-linked glycosylation site atresidues 26-28 of SEQ ID NO: 2. Furthermore, more than one region on theTNFR2-Fc polypeptide may be modified to introduce multiple N-linkedglycosylation sites including. For example, a double substitution ofD25N/E133N introduces two N-linked glycosylation sites at residues 25-27and 133-135 of SEQ ID NO: 2 and a double substitution of D25N/G231Nintroduces two N-linked glycosylation sites at residues 25-27 and231-233 of SEQ ID NO: 2. Other exemplary TNFR2 variants include, forexample, E133N/G231N, Q26N/A28S/E133N, Q26N/A28T/E133N andQ26N/A28S/G231N or Q26N/A28T/G231N. Any S that is predicted to beglycosylated may be altered to a T without creating an immunogenic site,due to the protection afforded by the glycosylation. Similarly, any Tthat is predicted to be glycosylated may be altered to an S.

In one embodiment, the glycovariant fusion proteins described hereincomprise a heterologous polypeptide portion that comprises anextracellular domain of a CTLA4 receptor or a ligand binding portionthereof. In exemplary embodiments, the fusion proteins described hereincomprise an extracellular domain of CTLA4 having at least onenon-endogenous N-linked glycosylation site.

As used herein, the term “CTLA4” refers to a family of CTLA4 proteinsfrom any species and variants derived from such CTLA4 proteins bymutagenesis or other modification. Members of the CTLA4 family aregenerally transmembrane proteins, composed of a ligand-bindingextracellular immunoglobulin V domain with a transmembrane domain, and acytoplasmic domain with predicted intracellular signaling activity.

The term “CTLA4 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of a CTLA4 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. For example, CTLA4polypeptides include polypeptides derived from the sequence of any knownCTLA4 having a sequence at least about 80% identical to the sequence ofa CTLA4 polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% orgreater identity. For example, a CTLA4 polypeptide of the invention maybind to CD80 or CD86. Examples of CTLA4 polypeptides include human CTLA4precursor polypeptide (SEQ ID NO:49) and soluble human CTLA4polypeptides (e.g., SEQ ID NOs: 49, 50 or 52.

The human CTLA4 precursor protein sequence is as follows (GenbankAF411058_(—)1):

(SEQ ID NO: 49)   1MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQPAVVLASS RGIASFVCEY  61ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR 121AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL 181LTAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN

The human CTLA4 soluble (extracellular), processed polypeptide sequenceis as follows:

(SEQ ID NO: 50) KAMHVAQPAV VLASSRGIAS FVCEYASPGK ATEVRVTVLRQADSQVTEVC AATYMMGNEL TFLDDSICTG TSSGNQVNLTIQGLRAMDTG LYICKVELMY PPPYYLGIGN GTQIYVIDPE PCPDSD

The nucleic acid sequence encoding human CTLA4 precursor protein is asfollows (Genbank entry AF414120):

(SEQ ID NO: 51) ATGGC TTGCCTTGGA TTTCAGCGGC ACAAGGCTCA GCTGAACCTGGCTACCAGGA CCTGGCCCTG CACTCTCCTG TTTTTTCTTCTCTTCATCCC TGTCTTCTGC AAAGCAATGC ACGTGGCCCAGCCTGCTGTG GTACTGGCCA GCAGCCGAGG CATCGCCAGCTTTGTGTGTG AGTATGCATC TCCAGGCAAA GCCACTGAGGTCCGGGTGAC AGTGCTTCGG CAGGCTGACA GCCAGGTGACTGAAGTCTGT GCGGCAACCT ACATGATGGG GAATGAGTTGACCTTCCTAG ATGATTCCAT CTGCACGGGC ACCTCCAGTGGAAATCAAGT GAACCTCACT ATCCAAGGAC TGAGGGCCATGGACACGGGA CTCTACATCT GCAAGGTGGA GCTCATGTACCCACCGCCAT ACTACCTGGG CATAGGCAAC GGAACCCAGATTTATGTAAT TGATCCAGAA CCGTGCCCAG ATTCTGACTTCCTCCTCTGG ATCCTTGCAG CAGTTAGTTC GGGGTTGTTTTTTTATAGCT TTCTCCTCAC AGCTGTTTCT TTGAGCAAAATGCTAAAGAA AAGAAGCCCT CTTACAACAG GGGTCTATGTGAAAATGCCC CCAACAGAGC CAGAATGTGA AAAGCAATTTCAGCCTTATT TTATTCCCAT CAATTGA.

The amino acid sequence for an example of a CTLA4-h(2)Fc fusion proteinis as follows:

(SEQ ID NO: 52) KAMHVAQPAV VLASSRGIAS FVCEYASPGK ATEVRVTVLRQADSQVTEVC AATYMMGNEL TFLDDSICTG TSSGNQVNLTIQGLRAMDTG LYICKVELMY PPPYYLGIGN GTQIYVIDPE PCPDSDTGGG THTCPPCPAP ELLGGPSVFL FPPKPKDTLMISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPREEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTVDKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK.

The addition of one or more N-linked glycosylation sites (NXS/T) to aCTLA4-Fc protein may increase the serum half-life relative an unmodifiedCTLA4-Fc protein. N-linked glycosylation sites may be introduced withminimal effort by introducing an N in the correct position with respectto a pre-existing S or T, or by introducing an S or T at a correctposition with respect to a pre-existing N. Particularly suitable sitesfor the introduction of non-endogenous NXS/T sequences in CTLA4 include,for example (numbering is in relation to SEQ ID NO:50), amino acids 9-11(AVV), 12-14 (LAS), 18-20 (IAS), 30-32 (KAT), 42-44 (ADS), 43-45 (DSQ),45-47 (QVT), 59-61 (ELT), 64-66 (DDS), 69-71 (TGT), 71-73 (TSS) and87-89 (MDT). These alterations may be introduced into any functionalCTLA4 ECD portion and fused to an appropriate Fc domain. For example,desirable alterations that would introduce an N-linked glycosylationsite are: A9N/V11S(or T), L12N, I18N, K30N, A42N, D43N/Q45S (or T),Q45N, E59N, D64N, T69N, T71N, and M87N. Any combination of the foregoingmay be made. Any S that is predicted to be glycosylated may be alteredto a T without creating an immunogenic site, due to the protectionafforded by the glycosylation. Similarly, any T that is predicted to beglycosylated may be altered to and S.

A variety of mutations in CTLA4 are known that alter its ligand bindingactivity. Any of these mutations may be combined with the foregoing andwith each other (again numbered per SEQ ID NO:50): L106E, A31Y, T32N,A52M, M56K, G57E, S66P, S72F.

3. Nucleic Acids Encoding Glycovariant Fusion Proteins

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the glycovariant fusion proteins of theinvention, including fragments and functional variants disclosed herein.The subject nucleic acids may be single-stranded or double-stranded.Such nucleic acids may be DNA or RNA molecules. These nucleic acids maybe used, for example, in methods for making glycovariant fusion proteinsof the invention or as direct therapeutic agents (e.g., in a genetherapy approach).

In exemplary embodiments, a nucleic acid of the invention encodes aTNFR2-Fc glycovariant fusion protein. For example, SEQ ID NO: 3 encodesthe naturally occurring human TNFR2 precursor polypeptides, while SEQ IDNO: 4 encodes the processed extracellular domain of TNFR2.

In certain embodiments, the subject nucleic acids encoding glycovariantfusion proteins of the invention are further understood to includenucleic acids that are variants of SEQ ID NO: 3, 4, 9, 17, 45 or 48.Variant nucleotide sequences include sequences that differ by one ormore nucleotide substitutions, additions or deletions, such as allelicvariants. In exemplary embodiments, the variant nucleic acids comprise anucleic acid sequence modification that results in the introduction ofone or more non-endogenous N-linked glycosylation sites in the encodedprotein.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 3, 4, 9, 17, 45 or 48, and includeone or more sequence modifications that result in the introduction of anon-endogenous N-linked glycosylation site in the encoded protein. Oneof ordinary skill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NO: 3, 4, 9, 17, 45 or 48, and variants of SEQID NO: 3, 4, 9, 17, 45 or 48 that introduce one or more non-endogenousN-linked glycosylation site in the encoded protein are also within thescope of this invention. In further embodiments, the nucleic acidsequences of the invention can be isolated, recombinant, and/or fusedwith a heterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 3, 4, 9, 17, 45 or 48,complement sequence of SEQ ID NO: 3, 4, 9, 17, 45 or 48, or fragmentsthereof. Such nucleotide sequences encode glycovariant protein fusionshaving one or more non-endogenous N-linked glycosylation sites. One ofordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Oneof ordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Forexample, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids variants which differ from the nucleic acids asset forth in SEQ ID NOs: 3, 4, 9, 17, 45 or 48 due to degeneracy in thegenetic code and that introduce one or more non-endogenous N-linkedglycosylation site in the encoded protein are also within the scope ofthe invention. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC are synonyms for histidine) mayresult in “silent” mutations which do not affect the amino acid sequenceof the protein. However, it is expected that DNA sequence polymorphismsthat do lead to changes in the amino acid sequences of the subjectproteins will exist among mammalian cells. One skilled in the art willappreciate that these variations in one or more nucleotides (up to about3-5% of the nucleotides) of the nucleic acids encoding a particularprotein may exist among individuals of a given species due to naturalallelic variation. Any and all such nucleotide variations and resultingamino acid polymorphisms are within the scope of this invention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain embodiments, the subject nucleic acid is provided in anexpression vector comprising a nucleotide sequence encoding aglycovariant fusion protein of the invention and operably linked to atleast one regulatory sequence. Regulatory sequences are art-recognizedand are selected to direct expression of a polypeptide of the invention.Accordingly, the term regulatory sequence includes promoters, enhancers,and other expression control elements. Exemplary regulatory sequencesare described in Goeddel; Gene Expression Technology: Methods inEnzymology, Academic Press, San Diego, Calif. (1990). For instance, anyof a wide variety of expression control sequences that control theexpression of a DNA sequence when operatively linked to it may be usedin these vectors to express DNA sequences encoding a glycovariant fusionprotein. Such useful expression control sequences, include, for example,the early and late promoters of SV40, tet promoter, adenovirus orcytomegalovirus immediate early promoter, RSV promoters, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage lambda, the control regions for fd coat protein, the promoterfor 3-phosphoglycerate kinase or other glycolytic enzymes, the promotersof acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other protein encoded by the vector, such as antibiotic markers,should also be considered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant glycovariant fusion protein of the invention includeplasmids and other vectors. For instance, suitable vectors includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant glycovariant fusion proteins by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject glycovariant fusion proteins of the invention in CHO cells,such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega,Madison, Wis.). As will be apparent, the subject gene constructs can beused to cause expression of the subject glycovariant fusion proteins incells propagated in culture, e.g., to produce proteins, including fusionproteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject glycovariant protein fusions of the invention. The host cell maybe any prokaryotic or eukaryotic cell. For example, a glycovariantfusion protein of the invention may be expressed in bacterial cells suchas E. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art. Preferably, the host cell will be a mammalian hostcell, such as a CHO or BHK cell line, that will produce a mammalianglycosylation pattern on the expressed protein.

Accordingly, the present invention further pertains to methods ofproducing the subject glycovariant fusion proteins of the invention. Forexample, a host cell transfected with an expression vector encoding anFc-fusion protein can be cultured under appropriate conditions to allowexpression of the Fc-fusion protein to occur. The Fc-fusion protein maybe secreted and isolated from a mixture of cells and medium containingthe Fc-fusion protein. Alternatively, the Fc-fusion protein may beretained cytoplasmically or in a membrane fraction and the cellsharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The subject Fc-fusion proteins can be isolatedfrom cell culture medium, host cells, or both, using techniques known inthe art for purifying proteins, including ion-exchange chromatography,gel filtration chromatography, ultrafiltration, electrophoresis,immunoaffinity purification with antibodies specific for particularepitopes of the Fc-fusion proteins and affinity purification with anagent that binds to a domain fused to the Fc-fusion protein (e.g., aprotein A column may be used to purify an Fc-fusion proteins). In apreferred embodiment, the Fc-fusion protein comprises an additionaldomain which facilitates its purification. In a preferred embodiment,purification is achieved by a series of column chromatography steps,including, for example, three or more of the following, in any order:protein A chromatography, Q sepharose chromatography, phenylsepharosechromatography, size exclusion chromatography, and cation exchangechromatography. The purification could be completed with viralfiltration and buffer exchange. Glycovariant fusion proteins of theinvention may be purified to a purity of >98% as determined by sizeexclusion chromatography and >95% as determined by SDS PAGE.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant glycovariantFc-fusion protein of the invention, can allow purification of theexpressed fusion protein by affinity chromatography using a Ni²⁺ metalresin. The purification leader sequence can then be subsequently removedby treatment with enterokinase to provide the purified glycovariantfusion protein of the invention (e.g., see Hochuli et al., (1987) J.Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Methods of Increasing Serum Half Life

In specific embodiments, the invention relates to methods for increasingthe stability and/or half-life (e.g. in vitro, in vivo, or serumhalf-life) of an Fc fusion protein by modifying the fusion protein tointroduce one or more non-endogenous N-linked glycosylation sitesoutside of the Fc domain. For example, such a method may includemodifying the sequence of an Fc fusion protein to introduce one or morenon-endogenous N-linked glycosylation site and expressing a nucleic acidencoding said modified polypeptide in a suitable cell, such as a Chinesehamster ovary (CHO) cell or a human cell to produce a glycovariant Fcfusion protein. Such a method may comprise: a) culturing a cell underconditions suitable for expression of the modified polypeptide, whereinsaid cell is transformed with a modified polypeptide expressionconstruct; and b) recovering the modified protein so expressed.Purification may be achieved by a series of purification steps,including for example, one, two, or three or more of the following, inany order: protein A chromatograph, anion exchange chromotography (e.g.,Q sepharose), hydrophobic interaction chromotography (e.g.,phenylsepharose), size exclusion chromatography, and cation exchangechromatography. Such polypeptide of the disclosure may be furtherformulated in liquid or solid (e.g., lyophilized forms). Any of theglycovariant fusion proteins described herein may be produced using thesaid method.

In preferred embodiments, glycosylation at one or more of the introducedglycosylations sites increases the half-life (e.g., in vitro, in vivo,serum half-life) of the modified fusion protein by at least 10%, 25%,50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, or 250% or more relativeto the serum half-life of the fusion protein lacking the introducedglycosylation site. In certain embodiments, the methods of thedisclosure may be used introduce at least one glycosylated amino acidper each 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, or 200 amino acids of fusion proteins of thedisclosure. In certain embodiments, the methods of the disclosure may beused introduce glycosylation sites so as to maintain a separationbetween each amino acid modified by an N-linked glycosylation, anO-linked glycosylation, or both, by at least 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 or more amino acids.

In certain embodiments, the disclosure provides methods for extendingthe half-life of a fusion protein comprising at least one heterologouspolypeptide domain and an immunoglobulin Fc domain. For example, suchmethods may include a) modifying an Fc-fusion protein outside of theimmunoglobulin Fc domain to introduce one or more non-endogenousN-linked glycosylation sites and b) expressing the Fc-fusion proteinsuch that one of the introduced glycosylation sites are glycosylated.The methods disclosed herein may be used, for example, to extend thehalf-life of an Fc-fusion protein comprising a portion of anextracellular domain (e.g., soluble portion) of a receptor that includesa ligand-binding domain. In certain embodiments, the methods are used tointroduce glycosylation sites outside of the ligand-binding domain ofthe Fc-fusion protein. In preferred embodiments, the methods forintroducing glycosylation sites do not significantly affect the bindingof the receptor portion of the fusion protein to the soluble ligand,e.g., ligand binding is affected by less than 2-, 3-, 5-, 10-, or15-fold relative to ligand binding to the Fc-fusion protein lacking theintroduced glycosylation site. In some embodiments, the methods of thedisclosure are used to introduce one or more glycosylation sites onfusion proteins comprising a linker domain. In certain embodiments, themethods of the disclosure may be used to introduce at least oneglycosylation site within the linker domain. Generally, the methodsdisclosed herein are used to increase the half-life of fusion proteinshaving heterologous domains that are of higher molecular weight, e.g.,proteins of higher molecular being at least 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 kDa.

5. Screening Assays

In certain aspects, the present invention provides screening assays foridentifying glycovariant Fc fusion proteins that maintain at least onebiological activity and have increased stability and/or increased serumhalf life. Glycovariant fusion proteins can be tested to assess theirability to modulate biological activities, to assess their stabilityand/or to assess their serum half life in vivo or in vitro. Glycovariantfusion proteins can be tested, for example, in animal models.

There are numerous approaches to screening for biological activity of aglycovariant fusion protein of the invention, for example, when assayinga glycovariant receptor-Fc fusion protein, the ability of the modifiedreceptor to bind to a ligand may be determined, or the ability of theglycovariant fusion protein to disrupt ligand-receptor signaling mayalso be assayed. In certain embodiments, high-throughput screening ofglycovariant fusion proteins can be carried out to identifyglycovariants that retain at least one biological activity of theunmodified protein, such as, for example, receptor variants that perturbligand or receptor-mediated effects on a selected cell line.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,glycovariant fusion proteins of the invention can be obtained byscreening polypeptides recombinantly produced from the correspondingfragment of the nucleic acid encoding said fusion proteins. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry.

Functionally active glycovariant fusion proteins of the invention can beobtained by screening libraries of modified fusion proteinsrecombinantly produced from the corresponding mutagenized nucleic acidsencoding said fusion proteins. The glycovariants can be produced andtested to identify those that retain at least biological activity of theunmodified fusion proteins, for example, the ability of a glycovariantreceptor fusion to act as an antagonist (inhibitor) of various cellularreceptor proteins (e.g., TNFR2 receptor proteins or CTLA4 proteins)and/or intracellular signaling mediated by a ligand-receptor binding.

Functional glycovariants may be generated by modifying the amino acidsequence of a fusion protein of the invention to increase stabilityand/or serum half life. Such modified glycovariant proteins whenselected to retain a biological activity, such as, for example, ligandbinding, are considered functional equivalents of the unmodified fusionproteins. Modified glycovariant fusion proteins can also be produced,for instance, by amino acid substitution, deletion, or addition tointroduce one or more N-linked glycosylation sites and/or other sequencemodifications, such as conservative substitutions. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of a fusion protein of the invention results ina functional homolog can be readily determined by assessing the abilityof the variant fusion protein to exhibit a biological activity in afashion similar to the unmodified fusion protein. For example, variantreceptor Fc fusion proteins, such as a TNFR2-Fc fusion protein orCTLA4-Fc fusion protein, may be screened for ability to bind to aspecific ligand (e.g., TNFα or TNFβ for TNFR2-Fc and CD80 or CD86 forCTLA4-Fc), to prevent binding of a ligand to a receptor polypeptide orto interfere with signaling caused by ligand binding to the receptor.CTLA4-Fc variant activity may also be measured by its ability to inhibitIL2 secretion by stimulated Jurkat human leukemic T cells. Linsley, P.S. et al. (1991) J. Exp. Med. 174:561.

A combinatorial library of glycovariant fusion proteins may be producedby way of a degenerate library of genes encoding a library of fusionproteins which each include at least a portion of potential polypeptidesequences. For instance, a mixture of synthetic oligonucleotides can beenzymatically ligated into gene sequences such that the degenerate setof potential polypeptide nucleotide sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display).

There are many ways by which the library of potential glycovariants canbe generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then be ligated intoan appropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam:Elsevier pp 273-289; Itakura et al., (1984) Annu Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic AcidRes. 11:477). Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al., (1990)Science 249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433;Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNASUSA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of glycovariant fusion proteins of theinvention. The most widely used techniques for screening large genelibraries typically comprises cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected.

Preferred assays include receptor-ligand binding assays andligand-mediated cell signaling assays. Complex formation betweenglycovariant receptor-Fc fusion proteins of the invention and ligandsmay be detected by a variety of techniques. For instance, modulation ofthe formation of complexes can be quantitated using, for example,detectably labeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or³H), fluorescently labeled (e.g., FITC), or enzymatically labeledreceptor polypeptide or ligands, by immunoassay, or by chromatographicdetection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between a glycovariant receptor-Fc fusion proteinand its binding ligand. Further, other modes of detection, such as thosebased on optical waveguides (PCT Publication WO 96/26432 and U.S. Pat.No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors,and surface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between a glycovariant Fc fusionprotein and its binding partner. See for example, U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) JBiol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696).Alternatively, such protein-protein interaction can be identified at theprotein level using in vitro biochemical methods, includingphoto-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, glycovariant fusion proteins may be screened in amechanism based assay, such as an assay to detect glycovariant fusionproteins which bind to a ligand or receptor polypeptide. This mayinclude a solid phase or fluid phase binding event. Alternatively, thegene encoding a ligand or receptor polypeptide can be transfected with areporter system (e.g., β-galactosidase, luciferase, or green fluorescentprotein) into a cell and screened against the glycovariant libraryoptionally by a high throughput screening or with individual members ofthe glycovariant library. Other mechanism based binding assays may beused, for example, binding assays which detect changes in free energy.Binding assays can be performed with the target fixed to a well, bead orchip or captured by an immobilized antibody or resolved by capillaryelectrophoresis. The bound proteins may be detected usually usingcolorimetric or fluorescence or surface plasmon resonance.

6. Exemplary Therapeutic Uses

In various embodiments, patients being treated with a glycovariantfusion protein of the invention, or candidate patients for treatmentwith a glycovariant fusion protein of the invention, may be mammals suchas rodents and primates, and particularly human patients. In certainembodiments, the disclosure provides modified soluble receptor-Fcfusions characterized by an increased half-life. Receptor-Fc fusionshave been previously described for use in treating a variety ofdisorders and conditions, which are summarized herein.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a therapeutic that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established. In eithercase, prevention or treatment may be discerned in the diagnosis providedby a physician or other health care provider and the intended result ofadministration of the therapeutic agent.

In some embodiments, the disclosure provides a glycovariant fusionprotein comprising an extracellular domain of a TNFR2 receptor having anincreased serum half life. Such TNFR2-Fc glycovariant fusions may beused to prevent or treat a variety of TNFα-mediated disorders orconditions including, for example acute and chronic immune andautoimmune pathologies (e.g., systemic lupus erythematosus (SLE),rheumatoid arthritis, juvenile chronic arthritis, thyroidosis, graftversus host disease, scleroderma, diabetes mellitus, Graves' disease),spondyloarthropathies, disorders associated with infection (e.g., sepsissyndrome, cachexia, circulatory collapse and shock resulting from acuteor chronic bacterial infection, acute and chronic parasitic and/orinfectious diseases, bacterial, viral or fungal), chronic inflammatorypathologies (e.g., scleroderma, sarcoidosis, chronic inflammatory boweldisease, ulcerative colitis, and Crohn's pathology), vascularinflammatory pathologies (e.g., disseminated intravascular coagulation,atherosclerosis, and Kawasaki's pathology), and neurodegenerativediseases (e.g., multiple sclerosis, acute transverse myelitis,extrapyramidal, Huntington's Chorea and senile chorea; Parkinson'sdisease, Progressive supranucleo palsy, spinal ataxia, Friedreich'sataxia, Mencel, Dejerine-Thomas, Shi-Drager, MachadoJoseph, Refsum'sdisease, abetalipoprotemia, telangiectasia, mitochondrial multi-systemdisorder, amyotrophic lateral sclerosis, infantile spinal muscularatrophy, juvenile spinal muscular atrophy, Alzheimer's disease, Down'sSyndrome, Diffuse Lewy body disease, Wernicke-Korsakoff syndrome,Creutzfeldt-Jakob disease, Subacute sclerosing panencephalitis,Hallerrorden-Spatz disease, and Dementia pugilistica).

In some embodiments, the disclosure provides a glycovariant fusionprotein comprising an extracellular domain of a CTLA4 receptor having anincreased serum half life. Such CTLA4-Fc glycovariant fusions may beused to prevent or treat a variety of CTLA4-related disorders. Giventhat both CTLA4 and TNFR2 participate in inflammation, the disordersthat are suitable for CTLA4-Fc proteins are similar to that for TNFR2-Fcfusion proteins including, for example acute and chronic immune andautoimmune pathologies (e.g., systemic lupus erythematosus (SLE),rheumatoid arthritis, juvenile chronic arthritis, thyroidosis, graftversus host disease, scleroderma, diabetes mellitus, Graves' disease) aswell treatment of patients who have received organ transplants,particularly those experiencing graft rejection.

7. Pharmaceutical Compositions

In certain embodiments, the glycovariant fusion proteins describedherein are formulated with a pharmaceutically acceptable carrier. Forexample, a fusion protein of the disclosure can be administered alone oras a component of a pharmaceutical formulation (therapeuticcomposition). The subject compounds may be formulated for administrationin any convenient way for use in human or veterinary medicine. Anappropriate dosage regimen may be determined by an attending physicianand may be informed by the standard dosage regimen for the unmodifiedversion of the therapeutic protein.

In certain embodiments, a therapeutic method of the invention includesadministering the composition systemically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the fusion proteinsdescribed herein which may optionally be included in the composition asdescribed above, may be administered simultaneously or sequentially withthe subject fusion proteins in the methods of the invention.

Typically, the fusion proteins described herein will be administeredparentally. Pharmaceutical compositions suitable for parenteraladministration may comprise one or more fusion proteins of thedisclosure in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site (e.g., bone, muscle, circulatorysystem, etc.). In certain embodiments, compositions of the presentinvention may include a matrix capable of delivering one or moretherapeutic compounds (e.g., glycovariant fusion proteins) to a targettissue site (e.g., bone, muscle, circulatory system), providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof a fusion protein of the disclosure. Such matrices may be formed ofmaterials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, fusion proteins can be formulated for oraladministration, e.g., in the form of capsules, cachets, pills, tablets,lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes and the like, each containing a predetermined amount ofan agent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, and powders granules), one or more fusion proteins ofthe present invention may be mixed with one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the fusion proteins, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 Expression of Glycovariant TNFR2-Fc Fusion Proteins

Applicants constructed vectors for expression of a variety of TNFR2-Fcfusion proteins, each containing an additional N-linked glycosylationsite. The control TNFR2-Fc fusion protein (i.e., the basic, or “initial”unmodified form of TNFR2-Fc) has the extracellular domain of human TNFR2fused to a human IgG1 Fc domain with no intervening linker. The sequenceof the initial TNFR2-Fc fusion protein is shown in FIG. 1 (SEQ ID NO:5),and the encoding nucleic acid (including the leader sequence) is shownin FIG. 5 (SEQ ID NO:8). The fusion protein shown in FIG. 1 is morefully designated TNFR2-h(1)Fc. A variant designated TNFR2-h(2)Fcpossesses an alternative linker/boundary sequence as shown in FIG. 9(SEQ ID NO:16) and is encoded by the nucleotide sequence indicated inFIG. 10 (SEQ ID NO: 17). Protein constructs were cloned into a pAID4vector for expression in mammalian cells (Pearsall et al. PNAS 105(2008): 7082-7087).

For initial experiments, glycovariants of TNFR2-Fc were transientlyexpressed in HEK293 cells. In brief, in a 500 ml spinner, HEK293T cellswere set up at 6×10⁵ cells/ml in Freestyle (Invitrogen) media in 250 mlvolume and grown overnight. Next day, these cells were treated withDNA:PEI (1:1) complex at 0.5 ug/ml final DNA concentration. After 4 hrs,250 ml media was added and cells were grown for 7 days. Conditionedmedia was harvested by spinning down the cells and concentrated.

Variants were purified using a variety of techniques, including, forexample, protein A column (Mab Select™, GE Healthcare LifeSciences, USA)and eluted with low pH (3.0) glycine buffer, followed by size exclusionchromatography. Proteins were dialyzed against phosphate buffered salineor Tris buffered saline.

While the protein sequences provided in FIG. 5 indicate an N-terminalsequence of “LPA . . . ”, a significant portion of each protein showedthat the N-terminal leucine had been removed, yielding an N-terminalsequence of “PAQ . . . ”. Thus any of the TNFR2-Fc molecules disclosedherein may have one or two amino acids removed from the N-terminus.

Three different leader sequences were considered for use:

-   -   (i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO:        10),    -   (ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP        (SEQ ID NO: 11), and    -   (iii) Native TNFR: MAPVAVWAALAVGLELWAAAHA (SEQ ID NO: 15).

Additional purification could be achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: Q sepharose chromatography, phenylsepharosechromatography, hydroxyapatite chromatography, and cation exchangechromatography. The purification could be completed with viralfiltration and buffer exchange.

Example 2 Design of Glycovariant TNFR2-Fc Fusion Proteins

Positions for introduction of additional N-linked glycosylation siteswere selected by inspection of the co-crystal structure of theextracellular domain of TNFR2 and its ligand TNF (crystal coordinatespublicly available), and by application of the principles describedherein. The alterations were chosen as being at positions predicted topreserve the TNF antagonist activity of the protein while conferring anextended half-life, and are shown in FIG. 7. Note that the numbering ofamino acids is based on the native, unprocessed TNFR2 amino acidsequence, as shown in FIG. 7. The altered TNFR2-Fc fusion proteins weredesigned to have an additional N-linked sugar moiety at one of thefollowing positions: 47, 48, 62, 114, 155, 202, 203, 222 and 253. Notethat the native TNFR2-Fc has N-linked sugar moieties at positions 171and 193, meaning that the heterologous domain of the initial TNFR2-Fcfusion protein has a ratio of N-linked sugar moieties to amino acids of1:117.5. The predicted positions of additional sugar moieties weremapped onto the TNFR2-TNF co-crystal structure and these are shown inFIG. 6. FIG. 8 indicates the predicted location of beta strand secondarystructure in the TNFR2 extracellular domain as inferred by sequencecomparison and structural homology with related proteins. Each of thenew N-linked sites were positioned internal to structural elements. Forexample, the sites introduced at positions 47 and 48 are internal to thefirst beta-sheet structural element. After generating and testingproteins with single additional N-linked glycosylation sites,combination mutants were generated and tested.

Example 3 Testing of TNFR2-Fc Glycovariant Proteins

Glycovariants of TNFR2-h(1)Fc were tested in a series of assays toassess functionality of the modified molecules and effects of themodification on pharmacokinetic properties of the molecules.

To assess ligand binding in a cell-free biochemical assay, a Biacore®3000 biosensor instrument was used. In brief, TNFR2-h(1)Fc variants wereloaded into the flow cell and then exposed to TNF. By evaluating kineticparameters (k_(a) and k_(d)), a dissociation constant (K_(D)) wascalculated.

To assess ligand inhibition, a cell-based assay for TNF signaling wasused, essentially as described in Khabar et al. Immunol. Lett. 46(1995): 107-110. In brief, WEHI cells (ATCC) were cultured in thepresence of TNF-alpha (R&D Systems, Minneapolis, Minn.) and ActinomycinD. TNF-alpha causes apoptosis in these cells, and lysis rate detected atA_(490nm). The presence of a TNF-alpha antagonist causes a decrease inthe signal which permits the detection of an IC50.

To assess pharmacokinetic properties of the molecules studies wereconducted in Sprague-Dawley rats using standard techniques. In brief,after an acclimation period, animals were dosed with a TNFR2-h(1)Fcmolecule and bled by tail-nick at hour 6, 10, 24, 32, 48, 72, 96, 144,216 and 312 after initial dose. The presence of TNFR2-Fc molecules wasdetected by an ELISA for the human Fc portion.

The data were normalized against data obtained from a nativeTNFR2-h(1)Fc molecule to minimize variability from experiment toexperiment. Normalized values are shown below. Proteins exhibitingdesirable qualities are shown in gray and underlined.

K_(D) IC50 T_(1/2) Protein Construct (TNF-alpha) (WEHI Assay) (Rat)TNFR-h(1)Fc wt  1.0   1.0   1.0 (valued normalized) D47N  1.6   1.9 Nd*Q48N A50S

H62N K64T No affinity Nd** Nd** Q114N R116S No affinity Nd** Nd** E155N

S202N

T203N P205S  0.33   0.29   0.44 T222N  0.78   0.25   0.45 G253N

D47N E155N  2.6   1.6   1.1 D47N S202N  4.4   1.1   1.1 D47N G253N 55  3.0   1.4 E155N G253N  3.6   0.57   0.72 S202N G253N  1.05   0.23  0.65 E155N S202N G253N  1.0   0.25   0.89 *D47N protein was notexpressed in sufficient quantity to permit testing in vivo. **Themutations placing the N-linked sugar at positions 62 or 114 causedcomplete disruption of ligand binding, and no other properties wereevaluated.

The data presented above indicate that nearly half of the variantscontaining a single additional N-linked site (4 of 9) exhibitedsubstantially improved serum half life while retaining ligand bindingand antagonism similar to that of the wild-type molecule. Because of theeffects of allometric scaling on serum half-life, changes in serumhalf-life of 20-30% in rats should provide a meaningful improvement inthe dosing regimen for human patients. Interestingly, combinationmutations generally seemed to either decrease serum half-life or proteinactivity, suggesting that, in this molecule, further increases in thedensity of glycosylation was no longer helpful.

These data demonstrate that, using the teachings provided herein, onecan efficiently generate Fc fusion molecules with extended serumhalf-lives.

Example 4 Pharmacokinetic Rat Assay

Pharmacokinetic properties of the glycovariant molecules may bedetermined in Sprague-Dawley rats using standard techniques. In brief,after an acclimation period, eight (8) animals are dosed subcutaneously(SC) with a glycovariant molecule and bled by tail-nick at hour 6, 10,24, 32, 48, 72, 96, 144, 216 and 312 after initial dose. The presence ofthe glycovariant molecules may be detected using any method suitable forthe target protein. For example, the glycovariant Fc fusion protein maybe detected using an ELISA for the human Fc portion. The serumelimination half-life (T_(1/2)) is calculated for each individualanimal. Mean T_(1/2) and standard deviations are calculated for theglycovariant molecule. If not already determined, similar studies may becarried out to determine the mean T_(1/2) and standard deviations for anappropriate control molecule administered at the same dose, such as anFc fusion protein that does not contain the introduced N-linkedglycosylation site. Mean T_(1/2) is analyzed using an appropriatestatistical test such as a Student's t-test. The mean T_(1/2) of theglycovariant molecule may be compared to the mean T_(1/2) of anappropriate control molecule.

A study was conducted according to the foregoing methods to comparepharmacokinetic properties of TNFR2-h(1)Fc, Q48N/A50S TNFR2-h(1)Fc, andQ48N/A50S TNFR2-h(2)Fc in rats. This study determined proteinconcentrations up to 28 days after subcutaneous administration of singledoses of 5 mg/kg (n=3 rats per construct).

Protein Construct T_(1/2) TNFR2-h(1)Fc (value normalized) 1.0 Q48N/A50STNFR2-h(1)Fc 1.5 Q48N/A50S TNFR2-h(2)Fc 1.8

Compared to TNFR2-h(1)Fc, serum half-lives of the glycovariantsQ48N/A50S TNFR2-h(1)Fc and Q48N/A50S TNFR2-h(2)Fc were found to belonger by 50% and 80%, respectively. Remarkably, the maximum serumconcentration (Cmax) for Q48N/A50S TNFR2-h(2)Fc was nearly five-foldhigher (20.8 μg/ml versus 4.4 μg/ml, p<0.05) than for Q48N/A50STNFR2-h(1)Fc. These results demonstrate that modification of thelinker/boundary region (compare SEQ ID NO: 16 with SEQ ID NO: 5) of aTNFR2-Fc glycovariant can endow that variant (Q48N/A50S TNFR2-h(2)Fc inthis example) with improved pharmacokinetic properties. Thus, thelinker-hFc sequence provided as SEQ ID NO:18 may be particularly usefulin combination with any of the glycovariants disclosed herein.

Example 5 Pharmacokinetic Monkey Assay

Pharmacokinetic properties of the glycovariant molecules may beconducted in cynomolgus monkey model using standard techniques. Inbrief, 4 male and 4 female cynomolgus monkeys are selected. The animalsare experimentally naive at the outset of the procedures, areapproximately 2 to 6 years of age, and are at least 2.5 kg in weight.Animals are fed a standard diet and housed according to standardprocedures throughout treatment. Animals are dosed with a glycovariantmolecule via subcutaneous (SC) or intravenous (IV) administration. Bloodsamples are collected via femoral venipuncture over a 22 day period atthe following timepoints post dosing: 5 minutes, 15 minutes, 30 minutes,1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 34 hours, 48hours, 58 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours,408 hours, and 504 hours. A predose blood sample is also collected.Following blood collection, the samples are stored on ice and the serumisolated by centrifugation. The presence of the glycovariant moleculesin the serum samples may be detected using any method suitable for thetarget protein. For example, the glycovariant Fc fusion protein may bedetected using an ELISA for the human Fc portion. The serum eliminationhalf-life (T_(1/2)) is calculated for each individual animal. MeanT_(1/2) and standard deviations are calculated for the glycovariantmolecule. If not already determined, similar studies may be carried outto determine the mean T_(1/2) and standard deviations for an appropriatecontrol molecule administered at the same dose, such as an Fc fusionprotein that does not contain the introduced N-linked glycosylationsite. Mean T_(1/2) is analyzed using an appropriate statistical testsuch as a Student's t-test. The mean T_(1/2) of the glycovariantmolecule may be compared to the mean T_(1/2) of an appropriate controlmolecule.

Example 6 Effect of Q48N/A50S TNFR2-h(1)Fc in a RatCollagen-Induced-Arthritis Model

The glycovariant Q48N/A50S TNFR2-h(1)Fc was evaluated for efficacyagainst collagen-induced arthritis in the rat. Bovine collagen type II(Elastin Products, catalog no. CN276) was dissolved in 0.01 M aceticacid and Freund's incomplete adjuvant (Difco, catalog no. 263910) to aconcentration of 1 mg/ml and was administered to female Lewis rats(150-200 g) by intradermal injection (2 mg/kg) at the base of the tailon study days 0 and 7. Starting on day 6, rats were treatedsubcutaneously three times per week with Q48N/A50S TNFR2-h(1)Fc at 3mg/kg, TNFR2-h(1)Fc at 3 mg/kg, or vehicle. Paw volume was determined byplethysmometry at baseline and multiple time points throughout thestudy, whereas bone quality was assessed by micro-computed tomography(micro-CT) at study conclusion (ex vivo).

Paw swelling was used as a marker for collagen-induced inflammation. Asexpected, paw volume in vehicle-treated rats increased midway throughthe study and remained elevated until the end. This paw swelling wasinhibited as effectively in rats treated with the glycovariant Q48N/A50STNFR2-h(1)Fc as it was in those treated with the positive control,TNFR2-h(1)Fc (FIG. 11). Moreover, bone quality as assessed by micro-CTwas improved similarly in both these treatment groups compared withvehicle-treated controls (FIG. 12), providing additional evidence ofanti-inflammatory efficacy. These data demonstrate that the glycovariantQ48N/A50S TNFR2-h(1)Fc has anti-inflammatory efficacy in vivo equivalentto that of its unsubstituted counterpart, TNFR2-h(1)Fc.

Taken together, the foregoing results indicate that certainglycovariants of TNFR2-h(1)Fc, for example Q48N/A50S TNFR2-h(1)Fc,possess increased serum half-life and undiminished anti-inflammatoryefficacy compared to TNFR2-h(1)Fc itself.

Example 7 Q48N/A50T, E155N TNFR2-Fc

Glycan analysis of the total sialic acid content was performed withrespect to the Q48N/A50S TNFR2-Fc fusion protein and the D47N/E155NTNFR2-Fc fusion protein. This assessment showed that the Q48N/A50Smutation introduced 195% more sialic acid relative to the wild-type formand the D47N/E155N mutation introduced 180% more sialic acid compared tothe wild-type TNFR2-Fc. This suggested that one or both of the D47N andE155N glycosylation sites is poorly sialylated. Detailed N-linked glycansize-based profiling (Amide-80 HPLC column) was used to assess therelative level of different N-glycans in TNFR2-Fc variants containingQ48N/A50S and D47N/E155N mutations. The analysis showed that theQ48N/A50S site is approximately 50% occupied with mono- anddi-sialylated glycan structures. The E155N site is >50% occupied with amix of sialylated and non-sialylated bi-antennary structures. The D47Nsite was occupied by Man5 and Man6 structures, and did not carry anysialic acid. This suggests that the Q48N/A50S and E155N sites yield adesirable increase in sialylation, while the D47N site creates anunsialylated glycoform that may have negative effects on serumhalf-life. Therefore a Q48N/A50S (or A50T) E155N TNFR2-Fc,particularly—h(2)Fc is expected to have a highly improved serumhalf-life. By comparing sequences of known N-linked glycosylation sitesthat have flanking regions similar to the those found in TNFR2, it wasdetermined that Thr is present in similar sequences at a greater ratethan Ser, and thus an N-glycosylation site created by Q48N/A50T mutationwould likely have higher probability of being fully glycosylated. AQ48N/A50T E155N TNFR2-h(2)Fc protein would have the followingunprocessed amino acid sequence:

(SEQ ID NO: 46) MAPVAVWAAL AVGLELWAAA HALPAQVAFT PYAPEPGSTC RLREYYDNTTQMCCSKCSPG QHAKVFCTKT SDTVCDSCEDSTYTQLWNWV PECLSCGSRC SSDQVETQAC TREQNRICTCRPGWYCALSK QEGCRLCAPL RKCRPGFGVA RPGTNTSDVVCKPCAPGTFS NTTSSTDICR PHQICNVVAI PGNASMDAVCTSTSPTRSMA PGAVHLPQPV STRSQHTQPT PEPSTAPSTSFLLPMGPSPP AEGSTGDTGG GTHTCPPCPA PELLGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDGVEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKCKVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKThe mature portion corresponding to TNFR2 is either 23-257 or 24-257depending on whether the L23 is retained.

Example 8 TNFR2-Fc Truncations

A series of C-terminal truncations of TNFR2-h(2)Fc were generated toassess the effects of including such truncations into a glycovariantprotein. Proteins were produced as described above and measured for IC50as described in Example 3, above.

TNFR2-Fc Constructs IC50 (ng/ml) WT · h(2)Fc 6.60 C′Δ21 · h(2)Fc 4.94C′Δ46 · h(2)Fc 10.41Additional C-terminal truncations of 26, 31, 36, 41, 54 and 76 aminoacids confirmed the decline in activity that is observed withtruncations deeper than 21 amino acids. Therefore, the form with 21amino acids deleted from the C-terminus (referred to as CΔ21) wasselected for further testing and for combination with glycovariants. Inparticular a Q48T/E155N CΔ21 TNFR2-h(2)Fc fusion protein was generated.The unprocessed amino acid sequence is as follows:

(SEQ ID NO: 47)   1MAPVAVWAAL AVGLELWAAA HALPAQVAFT PYAPEPGSTC RLREYYDNTT  51QMCCSKCSPG QHAKVFCTKT SDTVCDSCED STYTQLWNWV PECLSCGSRC 101SSDQVETQAC TREQNRICTC RPGWYCALSK QEGCRLCAPL RKCRPGFGVA 151RPGTNTSDVV CKPCAPGTFS NTTSSTDICR PHQICNVVAI PGNASMDAVC 201TSTSPTRSMA PGAVHLPQPV STRSQHTQPT PEPSTATGGG THTCPPCPAP 251ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV 301EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 351EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE 401SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 451HNHYTQKSLS LSPGK*The mature portion corresponding to TNFR2 is either 23-236 or 24-236depending on whether the L23 is retained.The protein is encoded by the following nucleic acid sequence:

(SEQ ID NO: 48)    1ATGGCGCCCG TCGCCGTCTG GGCCGCGCTG GCCGTCGGAC TGGAGCTCTG   51GGCTGCGGCG CACGCCTTGC CCGCCCAGGT GGCATTTACA CCCTACGCCC  101CGGAGCCCGG GAGCACATGC CGGCTCAGAG AATACTATGA CAACACAACC  151CAGATGTGCT GCAGCAAATG CTCGCCGGGC CAACATGCAA AAGTCTTCTG  201TACCAAGACC TCGGACACCG TGTGTGACTC CTGTGAGGAC AGCACATACA  251CCCAGCTCTG GAACTGGGTT CCCGAGTGCT TGAGCTGTGG CTCCCGCTGT  301AGCTCTGACC AGGTGGAAAC TCAAGCCTGC ACTCGGGAAC AGAACCGCAT  351CTGCACCTGC AGGCCCGGCT GGTACTGCGC GCTGAGCAAG CAGGAGGGGT  401GCCGGCTGTG CGCGCCGCTG CGCAAGTGCC GCCCGGGCTT CGGCGTGGCC  451AGACCAGGAA CTAACACATC AGACGTGGTG TGCAAGCCCT GTGCCCCGGG  501GACGTTCTCC AACACGACTT CATCCACGGA TATTTGCAGG CCCCACCAGA  551TCTGTAACGT GGTGGCCATC CCTGGGAATG CAAGCATGGA TGCAGTCTGC  601ACGTCCACGT CCCCCACCCG GAGTATGGCC CCAGGGGCAG TACACTTACC  651CCAGCCAGTG TCCACACGAT CCCAACACAC GCAGCCAACT CCAGAACCCA  701GCACTGCTAC CGGTGGTGGA ACTCACACAT GCCCACCGTG CCCAGCACCT  751GAACTCCTGG GGGGACCGTC AGTCTTCCTC TTCCCCCCAA AACCCAAGGA  801CACCCTCATG ATCTCCCGGA CCCCTGAGGT CACATGCGTG GTGGTGGACG  851TGAGCCACGA AGACCCTGAG GTCAAGTTCA ACTGGTACGT GGACGGCGTG  901GAGGTGCATA ATGCCAAGAC AAAGCCGCGG GAGGAGCAGT ACAACAGCAC  951GTACCGTGTG GTCAGCGTCC TCACCGTCCT GCACCAGGAC TGGCTGAATG 1001GCAAGGAGTA CAACTACAAG GTCTCCAACA AAGCCCTCCC AGCCCCCATC 1051GAGAAAACCA TCTCCAAAGC CAAAGGGCAG CCCCGAGAAC CACAGGTGTA 1101CACCCTGCCC CCATCCCGGG AGGAGATGAC CAAGAACCAG GTCAGCCTGA 1151CCTGCCTGGT CAAAGGCTTC TATCCCAGCG ACATCGCCGT GGAGTGGGAG 1201AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCACGCCTC CCGTGCTGGA 1251CTCCGACGGC TCCTTCTTCC TCTATAGCAA GCTCACCGTG GACAAGAGCA 1301GGTGGCAGCA GGGGAACGTC TTCTCATGCT CCGTGATGCA TGAGGCTCTG 1351CACAACCACT ACACGCAGAA GAGCCTCTCC CTGTCTCCGG GTAAATGAThis experiment indicates that deletion of amino acids at the N- orC-terminus from unstructured portions of the hyperglycosylated proteinmay provide useful benefits in the generation of glycovariants withextended serum half-life.

A further construct was generated incorporating the alterationsQ26N/A28T/E133N (each numbered as against the processed form) in the21-amino acid C-terminal deletion construct, thus providing a processedTNFR extracellular domain with the following sequence (modified aminoacids are in bold and italic, with new glycosylation sites underlined):

(SEQ ID NO: 53) LPAQVAFTPY APEPGSTCRL REYYD

QM CCSKCSPGQH AKVFCTKTSD TVCDSCEDST YTQLWNWVPE CLSCGSRCSSDQVETQACTR EQNRICTCRP GWYCALSKQE GCRLCAPLRK CRPGFGVARP GT

DVVCK PCAPGTFSNT TSSTDICRPH QICNVVAIPG NASMDAVCTS TSPTRSMAPG AVHLPQPVSTRSQHTQPTPE PSTAWhen incorporated into a fusion protein with an Fc domain and a linker,the resultant modified TNFR-Fc fusion protein retained excellent TNFbinding properties and provided superior homogeneity of expressedspecies compared to other glycoforms described in these examples. Anexample of an Fc fusion protein (with truncated hinge region) is shownbelow. Other Fc fusion protein forms could also be generated. The linkeris double underlined. The new glycosylation sites and the Fc region aresingle underlined.

(SEQ ID NO: 54) LPAQVAFTPY APEPGSTCRL REYYD

QM CCSKCSPGQH AKVFCTKTSD TVCDSCEDST YTQLWNWVPE CLSCGSRCSSDQVETQACTR EQNRICTCRP GWYCALSKQE GCRLCAPLRK CRPGFGVARP GT

DVVCK PCAPGTFSNT TSSTDICRPH QICNVVAIPG NASMDAVCTS TSPTRSMAPG AVHLPQPVSTRSQHTQPTPE PSTATGGGTH TCPPCPAPEL LGGPSVFLFPPKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEVHNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVSNKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVSLTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSFFLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A polypeptide comprising an amino acid sequence that is at least 99%identical to the amino acid sequence of SEQ ID NO:
 47. 2. Thepolypeptide of claim 1, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO:47.
 3. The polypeptide of claim 1, wherein thepolypeptide further comprises a constant domain of an immunoglobulinheavy chain.
 4. The polypeptide of claim 3, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:48.
 5. A polypeptidecomprising an amino acid sequence that is at least 99% identical to theamino acid sequence of SEQ ID NO:48.
 6. A fusion protein comprising animmunoglobulin Fc domain and at least one heterologous polypeptidedomain, wherein the heterologous polypeptide domain has an amino acidsequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical toSEQ ID NO:38, wherein the fusion protein is modified outside of theimmunoglobulin Fc domain to introduce at least one non-endogenousN-linked glycosylation site, and wherein glycosylation at the one ormore introduced glycosylation sites increases the serum half-life of themodified fusion protein by at least 10% relative to the serum half-lifeof the fusion protein lacking an introduced glycosylation site asmeasured in a pharmacokinetic monkey assay.
 7. The fusion protein ofclaim 6, wherein the glycosylation site is introduced in theextracellular domain outside of the ligand binding pocket.
 8. The fusionprotein of claim 6, wherein glycosylation at the one or more introducedglycosylation sites does not affect ligand binding activity of thereceptor by more than 3-fold.
 9. The fusion protein of claim 6, whereinthe fusion protein is modified by addition or deletion of at least oneamino acid residue to introduce at least one glycosylation site.
 10. Thefusion protein of claim 6, wherein the fusion protein is modified bysubstitution of at least one amino acid residue to introduce at leastone glycosylation site.
 11. The fusion protein of claim 6, whereinglycosylation at one or more of the introduced glycosylation sitesincrease the serum half-life of the fusion protein by at least 20%relative to the serum half-life of the fusion protein lacking anintroduced glycosylation site.
 12. The fusion protein of claim 6,wherein the modified heterologous polypeptide comprises at least oneN-linked glycosylation site per each 90 amino acids.
 13. The fusionprotein of claim 6, wherein the modified heterologous polypeptidecomprises at least one N-linked glycosylation site per each 65 aminoacids.
 14. The fusion protein of claim 6, wherein the fusion proteinfurther comprises a polypeptide linker between the Fc domain and theheterologous polypeptide domain.
 15. The fusion protein of claim 6,wherein at least one of the introduced glycosylation sites is located inthe linker.
 16. The fusion protein of claim 6, wherein the amino acidsequence that is at least 90% identical to SEQ ID NO:38 comprises anamino acid substitution at one or more amino acid residues selected fromQ26, D25, A28, E133, or G231 of SEQ ID NO:
 38. 17. The fusion protein ofclaim 16, wherein the amino acid sequence that is at least 90% identicalto SEQ ID NO:38 comprises a Q to N substitution at amino acid 26 of SEQID NO:38 and an A to S substitution at amino acid 28 of SEQ ID NO: 38.18. The fusion protein of claim 16, wherein the amino acid sequence thatis at least 90% identical to SEQ ID NO:38 comprises a Q to Nsubstitution at amino acid 26 of SEQ ID NO:38 and an A to T substitutionat amino acid 28 of SEQ ID NO:
 38. 19. The fusion protein of claim 16,wherein the amino acid sequence that is at least 90% identical to SEQ IDNO:38 comprises a Q to N substitution at amino acid 26 of SEQ ID NO:38,an A to T substitution at amino acid 28 of SEQ ID NO:38 and an E to Nsubstitution at amino acid 231 of SEQ ID NO:38.
 20. The fusion proteinof claim 16, wherein the amino acid sequence that is at least 90%identical to SEQ ID NO:38 comprises a Q to N substitution at amino acid26 of SEQ ID NO:38, an A to S substitution at amino acid 28 of SEQ IDNO:38 and an E to N substitution at amino acid 231 of SEQ ID NO:38. 21.The fusion protein of claim 16, wherein the amino acid sequence that isat least 90% identical to SEQ ID NO:38 comprises a D to N substitutionat amino acid 25 of SEQ ID NO: 38 and an E to N substitution at aminoacid 133 of SEQ ID NO:
 38. 22. The fusion protein of claim 16, whereinthe amino acid sequence that is at least 90% identical to SEQ ID NO:38comprises a D to N substitution at amino acid 25 of SEQ ID NO:38 and anG to N substitution at amino acid 231 of SEQ ID NO:
 38. 23. The fusionprotein of claim 6, wherein the fusion protein comprises a linker and Fcportion having the amino acid sequence of SEQ ID NO:
 18. 24. Apharmaceutical preparation comprising the fusion protein of claim 6 anda pharmaceutically acceptable carrier, wherein the preparation issubstantially free of pyrogenic materials so as to be suitable foradministration to a mammal. 25-96. (canceled)